Compounds and methods for modulating tmprss6 expression

ABSTRACT

Disclosed herein are compositions and compounds comprising modified oligonucleotides for modulating TMPRSS6 and modulating an iron accumulation disease, disorder and/or condition in an individual in need thereof. Iron accumulation diseases in an individual such as polycythemia, hemochromatosis or β-thalassemia can be treated, ameliorated, delayed or prevented with the administration of antisense compounds targeted to TMPRSS6.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0271USC2SEQ_ST25.txt created Sep. 20, 2021, which is 148 kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides methods, compounds, and compositions formodulating TMPRSS6 expression for the purpose of reducing ironaccumulation in an animal.

BACKGROUND OF THE INVENTION

Maintenance of iron balance in human beings is delicate because of thelimited capacity of the human physiology for iron absorption andexcretion (Finch, C. A. and Huebers, H. N. Engl. J. Med. 1982. 306:1520-1528). Iron deficiency is a widespread disorder and results fromany condition in which dietary iron intake does not meet the body'sdemands. Often, pathological blood loss contributes to negative ironbalance. Iron overload is also a common condition, and may result from agenetic cause, for example, mutations of different genes of ironmetabolism (Camaschella, C. Blood. 2005. 106: 3710-3717). The hepaticpeptide hormone, hepcidin plays a key role in body iron metabolism as itcontrols iron absorption and recycling (Ganz, T. Am. Soc. Hematol. Educ.Program 2006.507: 29-35; Kemna, E. H. et al., Clin. Chem. 2007. 53:620-628). Several proteins, including HFE (hemochromatosis protein)(Ahmad, K. A. et al., Blood Cells Mol Dis. 2002. 29: 361), transferrinreceptor 2 (Kawabata, H. et al., Blood 2005. 105: 376), and hemojuvelin(Papanikolaou, G. et al., Nat. Genet. 2004. 36: 77) also regulate thebody's iron levels.

Transmembrane protease, serine 6 (TMPRSS6) is a type II transmembraneserine protease and is expressed primarily in the liver (Velasco, G. etal., J. Biol. Chem. 2002. 277: 37637-37646). Mutations in TMPRSS6 havebeen implicated in iron deficiency anemia (Finberg, K. E. et al., Nat.Genet. 2008. 40: 569-571), where the level of hepcidin was found to beunusually elevated. A study of a human population with microcytic anemiafound that loss-of-function mutations in the TMPRSS6 gene lead tooverproduction of hepcidin, which, in turn, lead to defective ironabsorption and utilization (Melis, M. A. et al., Hematologica 2008. 93:1473-1479). TMPRSS6 participates in a transmembrane signaling pathwaytriggered by iron deficiency and suppresses diverse pathways of Hampactivation, the gene that encodes hepcidin (Du, X. et al., Science 2008.320: 1088-1092). Heterozygous loss of TMPRSS6 in HFE^(−/−) mice reducessystemic iron overload, while homozygous loss of TMPRSS6 in HFE^(−/−)mice causes systemic iron deficiency and elevated hepatic expression ofhepcidin (Finberg, K. E. et al., Blood 2011. 117: 4590-4599).

An example of an iron overload disorder is Hemochromatosis.Hemochromatosis (e.g. hemochromatosis type 1 or hereditaryhemochromatosis) is a disorder that results in excess intestinalabsorption of dietary iron from the gastrointestinal tract (Allen, K. J.et al., N. Engl. J. Med. 2008. 358: 221-230). This results in apathological increase in total body iron stores. Excess iron accumulatesin tissues and organs, particularly the liver, adrenal glands, heart,skin, gonads, joints and pancreas, and disrupt their normal function.Secondary complications, such as cirrhosis (Ramm, G. A. and Ruddell, R.G. Semin. Liver Dis. 2010. 30: 271-287), polyarthropathy (Carroll, G. J.et al., Arthritis Rheum. 2011. 63: 286-294), adrenal insufficiency,heart failure and diabetes (Huang, J. et al., Diabetes 2011. 60: 80-87)are common. Another example of an iron overload disorder isβ-thalassemia, where patients can develop iron overload caused byineffective erythropoiesis or transfusions to treat β-thalassemia.

To date, therapeutic strategies to treat iron overload disorders havebeen limited. Nucleic acid inhibitors such as siRNA and antisenseoligonucleotides have been suggested or developed, but none of thecompounds directly targeting TMPRSS6 (PCT Publications WO2014/076195,WO2012/135246, WO2014/190157, WO2005/0032733, WO 2013/070786 andWO2013/173635; U.S. Pat. No. 8,090,542; Schmidt et al. Blood. 2013,121(7):1200-8) have been approved for treating iron overload disorders.Accordingly, there is an unmet need for highly potent and tolerablecompounds to inhibit TMPRSS6. The invention disclosed herein relates tothe discovery of novel, highly potent inhibitors of TMPRSS6 expressionand their use in treatment.

All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

SUMMARY OF THE INVENTION

Provided herein are compositions, compounds and methods for modulatingthe levels of TMPRSS6 mRNA and/or protein in an animal. Provided hereinare compositions, compounds and methods for lowering TMPRSS6 levels.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide targeting a nucleic acid sequence encodingTMPRSS6. In certain embodiments, the compound targets a TMPRSS6 sequenceas shown in the nucleobase sequences of any of SEQ ID NOs: 1-6.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 3162 to 3184 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 1.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8 contiguousnucleobases of any of the nucleobase sequences of SEQ ID NOs: 23, 36,37, 63, 77.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide with the following formula:

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide with the following formula:

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout the disclosure herein are incorporated byreference for the portions of the document discussed herein, as well asin their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the 2′ position of a furosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“About” means within 10% of a value. For example, if it is stated, “amarker may be increased by about 50%”, it is implied that the marker maybe increased between 45%-55%.

“Active pharmaceutical agent” or “Pharmaceutical agent” means thesubstance or substances in a pharmaceutical composition that provide atherapeutic benefit when administered to an individual. For example, incertain embodiments, an antisense oligonucleotide targeted to TMPRSS6 isan active pharmaceutical agent.

“Active target region” or“target region” means a region to which one ormore active antisense compounds is targeted.

“Active antisense compounds” means antisense compounds that reducetarget nucleic acid levels or protein levels.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient time. Concomitant administration does notrequire that both agents be administered in a single pharmaceuticalcomposition, in the same dosage form, or by the same route ofadministration. The effects of both agents need not manifest themselvesat the same time. The effects need only be overlapping for a period oftime and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an individual,and includes, but is not limited to administering by a medicalprofessional and self-administering.

“Agent” means an active substance that can provide a therapeutic benefitwhen administered to an animal. “First Agent” means a therapeuticcompound provided herein. For example, a first agent is an antisenseoligonucleotide targeting TMPRSS6. “Second agent” means a secondtherapeutic compound described herein. For example, a second agent canbe a second antisense oligonucleotide targeting TMPRSS6 or a non-TMPRSS6target. Alternatively, a second agent can be a compound other than anantisense oligonucleotide.

“Amelioration” or “ameliorate” refers to a lessening of at least oneindicator, marker, sign, or symptom of an associated disease, disorderand/or condition. In certain embodiments, amelioration includes a delayor slowing in the progression of one or more indicators of a condition,disorder and/or disease. The severity of indicators may be determined bysubjective or objective measures, which are known to those skilled inthe art.

“Anemia” is a disease characterized by a lower than normal number of redblood cells (erythrocytes) in the blood, usually measured by a decreasein the amount of hemoglobin. The cause of anemia can include chronicinflammation, chronic kidney disease, kidney dialysis treatment, genetic(hereditary) disorders, chronic infection, acute infection, cancer andcancer treatments. Altered iron homeostasis and/or erythropoiesis inthese diseases, disorders and/or conditions can also result in decreasederythrocyte production. Clinical signs of anemia include low serum iron(hypoferremia), low hemoglobin levels, low hematocrit levels, decreasedred blood cells, decreased reticulocytes, increased soluble transferrinreceptor and iron restricted erythropoiesis. Examples of anemia includethalassemias (i.e. α-thalassemia, β-thalassemia (minor, intermedia andmajor) and δ-thalassemia), sickle cell anemia, aplastic anemia, Fanconianemia, Diamond Blackfan anemia, Shwachman Diamond syndrome, red cellmembrane disorders, glucose-6-phosphate dehydrogenase deficiency,hereditary hemorrhagic telangiectasia, hemolytic anemia, anemia ofchronic disease and the like.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specificallywith an antigen in some way, where the antibody and the antigen are eachdefined in terms of the other. Antibody may refer to a complete antibodymolecule or any fragment or region thereof, such as the heavy chain, thelight chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

“Antisense inhibition” means reduction of target nucleic acid levels ortarget protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

“Blood transfusion” refers to the process of receiving blood productsinto one's circulation intravenously. Transfusions are used in a varietyof medical disease, disorder and/or conditions to replace lost bloodcomponents.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising abridge connecting the 4′-carbon and the 2′-carbon, wherein the bridgehas the formula: 4′-CH(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleosidecomprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-0-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses concomitant, parallel orsequential administration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid. In certain embodiments,the first nucleic acid is an antisense compound and the second nucleicacid is a target nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′position of the sugar portion of the nucleotide. Deoxyribonucleotidesmay be modified with any of a variety of substituents.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition may be aliquid, e.g. phosphate buffered saline (PBS).

“Dosage unit” means a form in which a pharmaceutical agent is provided,e.g. pill, tablet, or other dosage unit known in the art. In certainembodiments, a dosage unit is a vial containing lyophilized antisenseoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted antisense oligonucleotide.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” or “therapeutically effective amount” means theamount of active pharmaceutical agent sufficient to effectuate a desiredphysiological outcome in an individual in need of the agent. Theeffective amount can vary among individuals depending on the health andphysical condition of the individual to be treated, the taxonomic groupof the individuals to be treated, the formulation of the composition,assessment of the individual's medical condition, and other relevantfactors.

“Fully complementary” or “100% complementary” means that each nucleobaseof a nucleobase sequence of a first nucleic acid has a complementarynucleobase in a second nucleobase sequence of a second nucleic acid. Incertain embodiments, the first nucleic acid is an antisense compound andthe second nucleic acid is a target nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap segment” andthe external regions may be referred to as “wing segments.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxynucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Hemochromatosis” is a disorder of iron metabolism that results inexcess iron being absorbed from the gastrointestinal tract, leading toexcess iron accumulation and deposition in various tissues of the body.Primary or hereditary or classic hemochromatosis is caused by a geneticmutation, for example, in the HFE gene. Subjects with this disease haveexcess amounts of iron, which is absorbed in the gastrointestinal tractand builds up in the body tissues, particularly in the liver. Secondaryor acquired hemochromatosis can be caused by frequent bloodtransfusions, high oral or parenteral intake of iron supplements, or asecondary effect of other diseases.

“Hematopoiesis” refers to the formation of cellular components of theblood, derived from hematopoietic stem cells. These stem cells reside inthe medulla of the bone marrow and have the unique ability to give riseto all the different mature blood cell types.

“Hemolysis” refers to the rupturing of erythrocytes or red blood cellsand the release of their contents into surrounding fluid. Hemolysis inan animal may occur due to a large number of medical conditions,including bacterial infection, parasitic infection, autoimmune disordersand genetic disorders.

“Hepcidin” refers to both an mRNA as well as a protein encoded by themRNA that is produced by hepatocytes in response to inflammation or torising levels of iron in the blood. The primary role of hepcidin is toregulate blood iron levels by facilitating a decrease in these bloodiron levels. Hepcidin expression is increased in conditions of acute andchronic inflammation resulting in decreased iron availability forerythropoiesis. “Hepcidin” is also referred to as hepcidin antimicrobialpeptide; HAMP; HAMP1; HEPC; HFE2; LEAP-1; LEAP1; and liver-expressedantimicrobial peptide.

“Hereditary anemia” refers to anemia which is caused by a hereditarycondition that causes red blood cells in the body to die faster thannormal, be ineffective in transporting oxygen from the lungs to thedifferent parts of the body, or not be created at all. Examples include,but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia,Diamond Blackfan anemia, Shwachman Diamond syndrome, red cell membranedisorders, glucose-6-phosphate dehydrogenase deficiency, or hereditaryhemorrhagic telangiectasia.

“HFE” refers to the human hemochromatosis gene or protein.

“HFE gene mutation” refers to mutations in the HFE gene, which mayresult in hereditary hemochromatosis.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Identifying an animal at risk for or having a disease, disorder and/orcondition associated with excess accumulation of iron” means identifyingan animal having been diagnosed with a disease, disorder and/orcondition or identifying an animal predisposed to develop a disease,disorder and/or condition associated with excess accumulation of iron.For example, an animal can be predisposed to develop a disease, disorderand/or condition associated with excess accumulation of iron if theanimal has a family history of hemochromatosis. Such identification maybe accomplished by any method including evaluating an animal's medicalhistory and standard clinical tests or assessments.

“Immediately adjacent” means that there are no intervening elementsbetween the immediately adjacent elements.

“Individual” or “subject” or “animal” means a human or non-human animalselected for treatment or therapy.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity of a RNA or protein and does notnecessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Intravenous administration” means administration into a vein.

“Iron accumulation” or “iron overload” indicates accumulation anddeposition of iron in the body from any cause. The most common causesare hereditary causes, transfusional iron overload, which can resultfrom repeated blood transfusions, or excessive dietary iron intake.

“Iron supplements” refer to supplements prescribed for a medical reasonto treat iron deficiency in a patient. Iron can be supplemented by theoral route or given parenterally.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Marker” or “biomarker” is any measurable and quantifiable biologicalparameter that serves as an index for health- or physiology-relatedassessments. For example, an increase in the percentage saturation oftransferrin, an increase in iron levels, or a decrease in hepcidinlevels can be considered markers of an iron overload disease, disorderand/or condition.

“MCH” refers to “mean corpuscular hemoglobin” or “mean cell hemoglobin”,a value to express the average mass of hemoglobin (Hb) per red bloodcell in a sample of blood.

“MCV” refers to “mean corpuscular volume” or “mean cell volume”, a valueto express the average red blood cell size.

“Mismatch” or “non-complementary nucleobase” or “MM” refers to the casewhen a nucleobase of a first nucleic acid is not capable of pairing withthe corresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e. aphosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. For example, a modifiednucleobase can be 5-methylcytosine. An “unmodified nucleobase” means thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, amodified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, and/or modifiednucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising amodified internucleoside linkage, a modified sugar, and/or a modifiednucleobase.

“Modified sugar” refers to a substitution or change from a naturalsugar. For example, a modified sugar can be 2′-MOE.

“Modulating” refers to changing or adjusting a feature in a cell,tissue, organ or organism. For example, modulating TMPRSS6 level canmean to increase or decrease the level of TMPRSS6 mRNA or TMPRSS6protein in a cell, tissue, organ or organism. A “modulator” effects thechange in the cell, tissue, organ or organism. For example, a TMPRSS6antisense oligonucleotide can be a modulator that increases or decreasesthe amount of TMPRSS6 mRNA or TMPRSS6 protein in a cell, tissue, organor organism.

“Monomer” refers to a single unit of an oligomer. Monomers include, butare not limited to, nucleosides and nucleotides, whether naturallyoccurring or modified.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“Mutations” refer to changes in a nucleic acid sequence. Mutations canbe caused in a variety of ways including, but not limited to, radiation,viruses, transposons and mutagenic chemicals, as well as errors thatoccur during meiosis, DNA replication, RNA transcription andpost-transcriptional processing. Mutations can result in severaldifferent changes in sequence; they can have either no effect, alter theproduct of a gene, or prevent the gene from functioning properly orcompletely. For example, HFE mutation can lead to the improperfunctioning of the gene product, leading to excess iron absorption inthe intestines.

“Myelodysplastic syndrome” refers to a diverse collection ofhematological medical disease, disorder and/or conditions that involveineffective production of the myeloid class of blood cells. The syndromeis caused by disorders of the stem cells in the bone marrow. Inmyelodysplastic syndrome, hematopoiesis is ineffective and the numberand quality of blood cells decline irreversibly, further impairing bloodproduction. As a result, patients with myelodysplastic syndrome developsevere anemia and require frequent blood transfusions.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids, double-stranded nucleic acids,small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound; such as, for example, nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Nucleotide mimetic” includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound; such as, for example, peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage).

“Oligomeric compound” or “oligomer” refers to a polymeric structurecomprising two or more sub-structures (monomers) and capable ofhybridizing to a region of a nucleic acid molecule. In certainembodiments, oligomeric compounds are oligonucleosides. In certainembodiments, oligomeric compounds are oligonucleotides. In certainembodiments, oligomeric compounds are antisense compounds. In certainembodiments, oligomeric compounds are antisense oligonucleotides. Incertain embodiments, oligomeric compounds are chimeric oligonucleotides.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intra-arterial administration, intraperitonealadministration, or intracranial administration, e.g., intrathecal orintracerebroventricular administration. Administration can becontinuous, or chronic, or short or intermittent.

“Peptide” refers to a molecule formed by linking at least two aminoacids by amide bonds. Peptide refers to polypeptides and proteins.

“Percentage saturation of transferrin” refers to the ratio of serum ironto total iron binding capacity multiplied by 100. Of the transferrinmolecules that are available to bind iron, this value tells a clinicianhow much serum iron are actually bound.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition may comprise one or more active pharmaceutical agents and asterile aqueous solution.

“Pharmaceutically acceptable carrier” means a medium or diluent thatdoes not interfere with the structure of the oligonucleotide. Certain ofsuch carriers enable pharmaceutical compositions to be formulated as,for example, tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspension and lozenges for the oral ingestion by a subject.For example, a pharmaceutically acceptable carrier can be a sterileaqueous solution, such as PBS.

“Pharmaceutically acceptable derivative” encompasses pharmaceuticallyacceptable salts, conjugates, prodrugs or isomers of the compoundsdescribed herein.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Polycythemia” refers to a condition of increased red blood cells (RBCs)in a specified volume due to either an increase in red blood cellnumbers (absolute polycythemia) or a decrease in plasma volume (relativepolycythemia). Blood volume to red blood cell proportions can bemeasured as Hematocrit (Hct) levels. The increased proportion of RBCscan make the blood viscous which can lead to slower blood flow throughthe circulatory system and potential formation of blood clots. Slowerblood flow can decrease oxygen transport to cells, tissue and/or organsleading to diseases, disorders or conditions such as angina or heartfailure. Formation of blood clots in the circulatory system can lead tocell, tissue and/or organ damage leading to diseases, disorders orconditions such as myocardial infarction or stroke. Treatment forpolycythemia includes phlebotomy or drugs to decrease RBC production(e.g., INF-α, hydroxyurea, anagrelide). Examples of polycythemiainclude, but is not limited to, polycythemia vera (PCV), polycythemiarubra vera (PRV) and erythremia. In certain instances, polycythemia canprogress into erythroid leukemia in a subject.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prevent” refers to delaying or forestalling the onset, development, orprogression of a disease, disorder, or condition for a period of timefrom minutes to indefinitely. Prevent also means reducing risk ofdeveloping a disease, disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Side effects” means physiological responses attributable to a treatmentother than the desired effects. In certain embodiments, side effectsinclude injection site reactions, liver function test abnormalities,renal function abnormalities, liver toxicity, renal toxicity, centralnervous system abnormalities, myopathies, and malaise. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity with a target nucleic acid toinduce a desired effect, while exhibiting minimal or no effects onnon-target nucleic acids under conditions in which specific binding isdesired, i.e. under physiological conditions in the case of in vivoassays and therapeutic treatments.

“Subcutaneous administration” means administration just below the skin.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Thalassemia” refers to a subgroup of anemias (e.g., α-thalassemia,β-thalassemia, δ-thalassemia, non-transfusion dependent thalassemia(NTDT)) caused by the formation of abnormal hemoglobin molecules leadingto the destruction or degradation of red blood cells. Complications ofthalassemia include excess iron (i.e. iron overload in the blood eitherfrom the thalassemia itself or from frequent transfusions to treat thethalassemia), increased risk of infection, bone deformities, enlargedspleens (i.e. splenomegaly), slowed growth rates and heart problems(e.g., congestive heart failure and arrhythmias).

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an animal.

“TMPRSS6” (also known as “matriptase-2”) refers to any nucleic acid orprotein of TMPRSS6.

“TMPRSS6 nucleic acid” means any nucleic acid encoding TMPRSS6. Forexample, in certain embodiments, a TMPRSS6 nucleic acid includes a DNAsequence encoding TMPRSS6, a RNA sequence transcribed from DNA encodingTMPRSS6 (including genomic DNA comprising introns and exons), and a mRNAsequence encoding TMPRSS6. “TMPRSS6 mRNA” means a mRNA encoding aTMPRSS6 protein.

“TMPRSS6 specific inhibitor” refers to any agent capable of specificallyinhibiting the expression of TMPRSS6 gene, TMPRSS6 RNA and/or TMPRSS6protein at the molecular level. For example, TMPRSS6 specific inhibitorsinclude nucleic acids (including antisense compounds), peptides,antibodies, small molecules, and other agents capable of inhibiting thelevel of TMPRSS6. In certain embodiments, by specifically modulatingTMPRSS6, TMPRSS6 specific inhibitors may affect components of the ironaccumulation pathway.

“Treat” refers to administering a pharmaceutical composition to ananimal in order to effect an alteration or improvement of a disease,disorder, or condition in the animal. In certain embodiments, one ormore pharmaceutical compositions can be administered to the animal.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleotide) or a DNA nucleotide (i.e. β-D-deoxyribonucleotide).

CERTAIN EMBODIMENTS

In certain embodiments disclosed herein, TMPRSS6 has the sequence as setforth in: GenBank Accession No. NM_153609.2 (incorporated herein as SEQID NO: 1); the complement of GENBANK Accession NT_011520.12 truncatedfrom 16850000 to U.S. Pat. No. 16,897,000 (incorporated herein as SEQ IDNO: 2); GENBANK Accession CR456446.1 (incorporated herein as SEQ ID NO:3); GENBANK Accession No. BC039082.1 (incorporated herein as SEQ ID NO:4); GENBANK Accession No. AY358398.1 (incorporated herein as SEQ ID NO:5); and GENBANK Accession No. DB081153.1 (incorporated herein as SEQ IDNO: 6).

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide targeting a nucleic acid sequence encodingTMPRSS6. In certain embodiments, the compound targets a TMPRSS6 sequenceas shown in the nucleobase sequences of any of SEQ ID NOs: 1-6.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising at least 8, least 9, least 10,least 11, at least 12, least 13, at least 14, at least 15, at least 16,least 17, least 18, least 19, or 20 contiguous nucleobases complementaryto an equal length portion of SEQ ID NOs: 1-6.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 3162 to 3184 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 1.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 1286 to 1305 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 1.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising a portion of at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, orat least 20 contiguous nucleobases complementary to an equal lengthportion of nucleobases 3162 to 3184 of SEQ ID NO: 1, wherein thenucleobase sequence of the modified oligonucleotide is at least 80%complementary to SEQ ID NO: 1.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising a portion of at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, orat least 20 contiguous nucleobases complementary to an equal lengthportion of nucleobases 1286 to 1305 of SEQ ID NO: 1, wherein thenucleobase sequence of the modified oligonucleotide is at least 80%complementary to SEQ ID NO: 1.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of any of the nucleobase sequences of SEQ ID NOs: 7-85.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of any of the nucleobase sequences of SEQ ID NOs: 23, 36,37, 63, 77.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of SEQ ID NO: 36.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosideshaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of SEQ ID NO: 77.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% complementary to an equal length portion of any ofSEQ ID NOs: 1-6. In certain embodiments, the modified oligonucleotidecomprises a nucleobase sequence 100% complementary to an equal lengthportion of any of SEQ ID NOs: 1-6.

In certain embodiments, the compound comprises a modifiedoligonucleotide consisting of 8 to 80, 20 to 80, 10 to 50, 20 to 35, 10to 30, 12 to 30, 15 to 30, 16 to 30, 20 to 30, 20 to 29, 20 to 28, 20 to27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 15 to25, 16 to 25, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 19 to22, 16 to 21, 18 to 21 or 16 to 20 linked nucleobases. In certainembodiments, the compound comprises a modified oligonucleotideconsisting of 16 linked nucleosides. In certain embodiments, thecompound comprises a modified oligonucleotide consisting of 20 linkednucleosides.

In certain embodiments, the compound comprises a modifiedoligonucleotide consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, or 80 linked nucleobases in length, or arange defined by any two of the above values.

In certain embodiments, the modified oligonucleotide is single-stranded.

In certain embodiments, the modified oligonucleotide comprises at leastone modified internucleoside linkage. In certain embodiments, themodified internucleoside linkage is a phosphorothioate internucleosidelinkage. In certain embodiments, at least one modified internucleosidelinkage is a phosphorothioate internucleoside linkage. In certainembodiments, each modified internucleoside linkage is a phosphorothioateinternucleoside linkage.

In certain embodiments, the modified oligonucleotide comprises at leastone nucleoside comprising a modified sugar. In certain embodiments, atleast one modified sugar comprises a bicyclic sugar. In certainembodiments, at least one modified sugar comprises a 2′-O-methoxyethyl,a constrained ethyl, a 3′-fluoro-HNA or a 4′-(CH₂)_(n)—O-2′ bridge,wherein n is 1 or 2.

In certain embodiments, the modified oligonucleotide comprises at leastone nucleoside comprising a modified nucleobase. In certain embodiments,the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises aconjugate group. In certain embodiments, the conjugate is a carbohydratemoiety. In certain embodiments, the conjugate is a GalNAc moiety. Incertain embodiments, the GalNAc is 5′-Trishexylamino-(THA)-C6 GalNAc₃.In certain embodiments, the conjugate has the formula

In certain embodiments, the compound comprises a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and targetedto or complementary to an equal length portion of region 3162 to 3184 ofSEQ ID NO: 1, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of linked deoxynucleosides; (b) a 5′ wing segmentconsisting of linked nucleosides; and (c) a 3′ wing segment consistingof linked nucleosides; wherein the gap segment is positioned immediatelyadjacent to and between the 5′ wing segment and the 3′ wing segment andwherein each nucleoside of each wing segment comprises a modified sugar.In certain embodiments, the modified oligonucleotide further comprisesat least one phosphorothioate internucleoside linkage. In certainembodiments, the modified oligonucleotide further comprises a GalNAcconjugate. In certain embodiments, the conjugate is a5′-Trishexylamino-(THA)-C6 GalNAc₃ conjugate.

In certain embodiments, the compound comprises a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and targetedto or complementary to an equal length portion of region 1286 to 1305 ofSEQ ID NO: 1, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of linked deoxynucleosides; (b) a 5′ wing segmentconsisting of linked nucleosides; and (c) a 3′ wing segment consistingof linked nucleosides; wherein the gap segment is positioned immediatelyadjacent to and between the 5′ wing segment and the 3′ wing segment andwherein each nucleoside of each wing segment comprises a modified sugar.In certain embodiments, the modified oligonucleotide further comprisesat least one phosphorothioate internucleoside linkage. In certainembodiments, the modified oligonucleotide further comprises a GalNAcconjugate. In certain embodiments, the conjugate is a5′-Trishexylamino-(THA)-C6 GalNAc₃ conjugate.

In certain embodiments, the compound comprises a modifiedoligonucleotide consisting of 20 linked nucleosides and targeted to orcomplementary to an equal length portion of region 3162 to 3181 of SEQID NO: 1, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; and (c) a 3′ wing segmentconsisting of five linked nucleosides; wherein the gap segment ispositioned immediately adjacent to and between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, wherein at least oneinternucleoside linkage is a phosphorothioate linkage and wherein eachcytosine residue is a 5-methylcytosine. In certain embodiments, themodified oligonucleotide further comprises a GalNAc conjugate. Incertain embodiments, the conjugate is a 5′-Trishexylamino-(THA)-C6GalNAc₃ conjugate.

In certain embodiments, the compound comprises a modifiedoligonucleotide consisting of 16 linked nucleosides and targeted to orcomplementary to an equal length portion of region 3169 to 3184 of SEQID NO: 1, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of nine linked deoxynucleosides; (b) a 5′ wingsegment consisting of three linked nucleosides; and (c) a 3′ wingsegment consisting of four linked nucleosides; wherein the gap segmentis positioned immediately adjacent to and between the 5′ wing segmentand the 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a modified sugar, wherein each internucleoside linkage is aphosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine. In certain embodiments, the modified oligonucleotidefurther comprises a GalNAc conjugate. In certain embodiments, theconjugate is a 5′-Trishexylamino-(THA)-C6 GalNAc₃ conjugate.

In certain embodiments, the compound comprising a modifiedoligonucleotide consisting of 20 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of SEQID NO: 36, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; and (c) a 3′ wing segmentconsisting of five linked nucleosides; wherein the gap segment ispositioned immediately adjacent to and between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, wherein at least oneinternucleoside linkage is a phosphorothioate linkage and wherein eachcytosine residue is a 5-methylcytosine. In certain embodiments, themodified oligonucleotide further comprises a GalNAc conjugate. Incertain embodiments, the conjugate is a 5′-Trishexylamino-(THA)-C6GalNAc₃ conjugate.

In certain embodiments, the compound comprising a modifiedoligonucleotide consisting of 16 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of SEQID NO: 77, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of nine linked deoxynucleosides; (b) a 5′ wingsegment consisting of three linked nucleosides; and (c) a 3′ wingsegment consisting of four linked nucleosides; wherein the gap segmentis positioned immediately adjacent to and between the 5′ wing segmentand the 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a modified sugar, wherein each internucleoside linkage is aphosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine. In certain embodiments, the modified oligonucleotidefurther comprises a GalNAc conjugate. In certain embodiments, theconjugate is a 5′-Trishexylamino-(THA)-C6 GalNAc₃ conjugate.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide according to the following formula: mCes TeoTeo Teo Aeo Tds Tds mCds mCds Ads Ads Ads Gds Gds Gds mCeo Aeo Ges mCesTe (SEQ ID NO: 36); wherein, A is an adenine, mC is a 5-methylcytosine,G is a guanine, T is a thymine, e is a 2′-O-methoxyethyl modifiednucleoside, d is a 2′-deoxynucleoside, and s is a phosphorothioateinternucleoside linkage. In certain embodiments, the modifiedoligonucleotide further comprises a GalNAc conjugate. In certainembodiments, the conjugate is a 5′-Trishexylamino-(THA)-C6 GalNAc₃conjugate.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide with the following formula:

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide according to the following formula: mCks AesGks mCds Tds Tds Tds Ads Tds Tds mCds mCds Aes Aes Aks Gk (SEQ ID NO:77); wherein, A is an adenine, mC is a 5-methylcytosine, G is a guanine,T is a thymine, e is a 2′-O-methoxyethyl modified nucleoside, d is a2′-deoxynucleoside, s is a phosphorothioate internucleoside linkage, andk is a cEt. In certain embodiments, the modified oligonucleotide furthercomprises a GalNAc conjugate. In certain embodiments, the conjugate is a5′-Trishexylamino-(THA)-C6 GalNAc₃ conjugate.

Certain embodiments disclosed herein provide a compound comprising amodified oligonucleotide with the following formula:

In certain embodiments, the compounds or compositions disclosed hereincomprise a salt of the modified oligonucleotide.

In certain embodiments, the compounds or compositions disclosed hereinfurther comprise a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the animal is a human.

Certain embodiments provide a composition or compound comprising amodified oligonucleotide as described herein, wherein the viscositylevel is less than 40 cP. In certain embodiments, the composition has aviscosity level less than 15 cP. In certain embodiments, the compositionhas a viscosity level less than 12 cP. In certain embodiments, thecomposition has a viscosity level less than 10 cP.

Certain embodiments disclosed herein provide compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 for use inreducing TMPRSS6 in a cell, tissue, organ or animal.

Certain embodiments disclosed herein provide compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 for use inreducing iron levels in a cell, tissue, organ or animal. In certainembodiments, the compounds and compositions reduce serum iron levels. Incertain embodiments, the compounds and compositions reduce liver ironlevels. In certain embodiments, the compounds and compositions reduceiron absorption. In certain embodiments, the compounds and compositionsreduce iron overload or accumulation. In certain embodiments, reducingiron overload/accumulation ameliorates, treats, prevents or delays adisease, disorder or condition related to iron overload.

Certain embodiments disclosed herein provide compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 for use inincreasing hepcidin levels, such as mRNA or protein expression levels,in an animal.

Certain embodiments disclosed herein provide compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 for use indecreasing the percentage saturation of transferrin in an animal. Incertain embodiments, decreasing transferrin saturation leads to adecrease in iron supply for erythropoiesis. In certain embodiments, thedecrease in erythropoiesis treats, prevents, delays the onset of,ameliorates, and/or reduces polycythemia, or symptom thereof, in theanimal. In certain embodiments, the polycythemia is polycythemia vera.In certain embodiments, treatment with the modified oligonucleotidetargeting TMPRSS6 prevents or delays the polycythemia from progressinginto erythroid leukemia.

Certain embodiments disclosed herein provide compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 for reducingiron accumulation in an animal. In certain embodiments, compounds andcompositions comprising a modified oligonucleotide targeting TMPRSS6 areused for treating, preventing, slowing the progression, delaying theonset of, ameliorating and/or reducing a disease, disorder and/orcondition, or symptom thereof, associated with the excess accumulationof iron in an animal.

In certain embodiments, the iron accumulation is the result of, or causeof, a disease, disorder or condition in the animal. In certainembodiments, the disease, disorder or condition is ineffectiveerythropoiesis, polycythemia, hemochromatosis or anemia. In certainembodiments, the hemochromatosis is hereditary hemochromatosis. Incertain embodiments, the anemia is hereditary anemia, myelodysplasticsyndrome or severe chronic hemolysis. In certain embodiments, thehereditary anemia is sickle cell anemia, thalassemia, Fanconi anemia,Diamond Blackfan anemia, Shwachman Diamond syndrome, red cell membranedisorders, glucose-6-phosphate dehydrogenase deficiency, or hereditaryhemorrhagic telangiectasia. In certain embodiments, the thalassemia isβ-thalassemia. In certain embodiments, the β-thalassemia isβ-thalassemia major, β-thalassemia intermedia or β-thalassemia minor. Incertain embodiments, the disease, disorder or condition is associatedwith mutations in the HFE gene. In other embodiments, the disease isassociated with mutations in the hemojuvelin gene. In other embodiments,the disease is associated with mutations in the hepcidin gene.

In certain embodiments, the iron accumulation is the result of a therapyto treat a disease, disorder or condition in the animal. In certainembodiments, the therapy is phlebotomy or transfusion therapy. Incertain embodiments, the disease, disorder and/or condition may be dueto multiple blood transfusions. In certain embodiments, multipletransfusions may lead to polycythemia. In certain embodiments, multipleblood transfusions are associated with the animal having anemia.Examples of anemia requiring multiple blood transfusions are hereditaryanemia, myelodysplastic syndrome and severe chronic hemolysis.

In certain embodiments, the disease, disorder and/or condition isassociated with excess parenteral iron supplement intake or excessdietary iron intake.

In certain embodiments, provided are compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 for use intherapy. In certain embodiments, the compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 are administeredto an animal in a therapeutically effective amount.

In certain embodiments, provided are compounds and compositionscomprising a modified oligonucleotide targeting TMPRSS6 for use in thepreparation of a medicament. In certain embodiments, the medicament isused for treating, preventing, slowing the progression, delaying theonset of, and/or reducing a disease, disorder and/or condition, orsymptom thereof, associated with excess accumulation of iron in ananimal.

In certain embodiments, the composition or compound comprising amodified oligonucleotide targeting TMPRSS6 is co-administered with oneor more second agent(s). In certain embodiments the second agent is aniron chelator or a hepcidin agonist. In further embodiments, the ironchelator includes FBS0701 (FerroKin), Exjade, Desferal or Deferiprone(DFP). In certain embodiments, the second agent is a second antisensecompound. In further embodiments, the second antisense compound targetsTMPRSS6. In other embodiments, the second antisense compound targets anon-TMPRSS6 compound. In other embodiments, the composition or compoundcomprising a modified oligonucleotide targeting TMPRSS6 is administeredbefore, during or after phlebotomy or transfusion therapy.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound can be “antisense” to a target nucleic acid, meaningthat it is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to TMPRSS6nucleic acid is 10 to 30 nucleotides in length. In other words,antisense compounds are from 10 to 30 linked nucleobases. In otherembodiments, the antisense compound comprises a modified oligonucleotideconsisting of 8 to 80, 10 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22,or 20 linked nucleobases. In certain such embodiments, the antisensecompound comprises a modified oligonucleotide consisting of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linkednucleobases in length, or a range defined by any two of the abovevalues. In some embodiments, the antisense compound is an antisenseoligonucleotide.

In certain embodiments, the antisense compound comprises a shortened ortruncated modified oligonucleotide. The shortened or truncated modifiedoligonucleotide can have a single nucleoside deleted from the 5′ end (5′truncation), the central portion or alternatively from the 3′ end (3′truncation). A shortened or truncated oligonucleotide can have two ormore nucleosides deleted from the 5′ end, two or more nucleosidesdeleted from the central portion or alternatively can have two or morenucleosides deleted from the 3′ end. Alternatively, the deletednucleosides can be dispersed throughout the modified oligonucleotide,for example, in an antisense compound having one or more nucleosidedeleted from the 5′ end, one or more nucleoside deleted from the centralportion and/or one or more nucleoside deleted from the 3′ end.

When a single additional nucleoside is present in a lengthenedoligonucleotide, the additional nucleoside can be located at the 5′ end,3′ end or central portion of the oligonucleotide. When two or moreadditional nucleosides are present, the added nucleosides can beadjacent to each other, for example, in an oligonucleotide having twonucleosides added to the 5′ end (5′ addition), to the 3′ end (3′addition) or the central portion, of the oligonucleotide. Alternatively,the added nucleoside can be dispersed throughout the antisense compound,for example, in an oligonucleotide having one or more nucleoside addedto the 5′ end, one or more nucleoside added to the 3′ end, and/or one ormore nucleoside added to the central portion.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modifiedsubunits arranged in patterns, or motifs, to confer to the antisensecompounds properties such as enhanced inhibitory activity, increasedbinding affinity for a target nucleic acid, or resistance to degradationby in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may confer another desired property e.g.,serve as a substrate for the cellular endonuclease RNase H, whichcleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving thehybridization of the antisense compound (e.g., oligonucleotide) with atarget nucleic acid, wherein the hybridization ultimately results in abiological effect. In certain embodiments, the amount and/or activity ofthe target nucleic acid is modulated. In certain embodiments, the amountand/or activity of the target nucleic acid is reduced. In certainembodiments, hybridization of the antisense compound to the targetnucleic acid ultimately results in target nucleic acid degradation. Incertain embodiments, hybridization of the antisense compound to thetarget nucleic acid does not result in target nucleic acid degradation.In certain such embodiments, the presence of the antisense compoundhybridized with the target nucleic acid (occupancy) results in amodulation of antisense activity. In certain embodiments, antisensecompounds having a particular chemical motif or pattern of chemicalmodifications are particularly suited to exploit one or more mechanisms.In certain embodiments, antisense compounds function through more thanone mechanism and/or through mechanisms that have not been elucidated.Accordingly, the antisense compounds described herein are not limited byparticular mechanism.

Antisense mechanisms include, without limitation, RNase H mediatedantisense; RNAi mechanisms, which utilize the RISC pathway and include,without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancybased mechanisms. Certain antisense compounds may act through more thanone such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part fromdegradation of target RNA by RNase H. RNase H is a cellular endonucleasethat cleaves the RNA strand of an RNA:DNA duplex. It is known in the artthat single-stranded antisense compounds which are “DNA-like” elicitRNase H activity in mammalian cells. Accordingly, antisense compoundscomprising at least a portion of DNA or DNA-like nucleosides mayactivate RNase H, resulting in cleavage of the target nucleic acid. Incertain embodiments, antisense compounds that utilize RNase H compriseone or more modified nucleosides. In certain embodiments, such antisensecompounds comprise at least one block of 1-8 modified nucleosides. Incertain such embodiments, the modified nucleosides do not support RNaseH activity. In certain embodiments, such antisense compounds aregapmers, as described herein. In certain such embodiments, the gap ofthe gapmer comprises DNA nucleosides. In certain such embodiments, thegap of the gapmer comprises DNA-like nucleosides. In certain suchembodiments, the gap of the gapmer comprises DNA nucleosides andDNA-like nucleosides.

Certain antisense compounds having a gapmer motif are consideredchimeric antisense compounds. In a gapmer an internal region having aplurality of nucleotides that supports RNaseH cleavage is positionedbetween external regions having a plurality of nucleotides that arechemically distinct from the nucleosides of the internal region. In thecase of an antisense oligonucleotide having a gapmer motif, the gapsegment generally serves as the substrate for endonuclease cleavage,while the wing segments comprise modified nucleosides. In certainembodiments, the regions of a gapmer are differentiated by the types ofsugar moieties comprising each distinct region. The types of sugarmoieties that are used to differentiate the regions of a gapmer may insome embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides,2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOEand 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides(such bicyclic sugar modified nucleosides may include those having aconstrained ethyl). In certain embodiments, nucleosides in the wings mayinclude several modified sugar moieties, including, for example 2′-MOEand bicyclic sugar moieties such as constrained ethyl (cEt) or LNA. Incertain embodiments, wings may include several modified and unmodifiedsugar moieties. In certain embodiments, wings may include variouscombinations of 2′-MOE nucleosides, bicyclic sugar moieties such asconstrained ethyl nucleosides or LNA nucleosides, and2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, oralternating sugar moieties. The wing-gap-wing motif is frequentlydescribed as “X-Y-Z”, where “X” represents the length of the 5′-wing,“Y” represents the length of the gap, and “Z” represents the length ofthe 3′-wing. “X” and “Z” may comprise uniform, variant, or alternatingsugar moieties. In certain embodiments, “X” and “Y” may include one ormore 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As usedherein, a gapmer described as “X-Y-Z” has a configuration such that thegap is positioned immediately adjacent to each of the 5′-wing and the 3′wing. Thus, no intervening nucleotides exist between the 5′-wing andgap, or the gap and the 3′-wing. Any of the antisense compoundsdescribed herein can have a gapmer motif. In certain embodiments, “X”and “Z” are the same; in other embodiments they are different. Incertain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z canbe any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30 or more nucleosides.

In certain embodiments, the antisense compound targeted to a TMPRSS6nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motifdescribed by Formula A as follows:(J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; xis 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNAcompounds (RNAi), which include double-stranded RNA compounds (alsoreferred to as short-interfering RNA or siRNA) and single-stranded RNAicompounds (or ssRNA). Such compounds work at least in part through theRISC pathway to degrade and/or sequester a target nucleic acid (thus,include microRNA/microRNA-mimic compounds). In certain embodiments,antisense compounds comprise modifications that make them particularlysuited for such mechanisms.

i. ssRNA compounds

In certain embodiments, antisense compounds including those particularlysuited for use as single-stranded RNAi compounds (ssRNA) comprise amodified 5′-terminal end. In certain such embodiments, the 5′-terminalend comprises a modified phosphate moiety. In certain embodiments, suchmodified phosphate is stabilized (e.g., resistant todegradation/cleavage compared to unmodified 5′-phosphate). In certainembodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorousmoiety. Certain modified 5′-terminal nucleosides may be found in theart, for example in WO 2011/139702.

In certain embodiments, the 5′-nucleoside of an ssRNA compound hasFormula IIc:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of FormulaIIc to the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄ R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇),OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl,C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; R₁₅, R₁₆, R₁₇ and R₁₈ areeach, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl,C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

Bx₁ is a heterocyclic base moiety;

or if Bx₂ is present then Bx₂ is a heterocyclic base moiety and Bx₁ isH, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl,C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

or J₄ forms a bridge with one of J₅ or J₇ wherein said bridge comprisesfrom 1 to 3 linked biradical groups selected from O, S, NR₁₉,C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other twoof J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E2)(E3);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, N(J₁)(J₂),=NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomericsubunits and is hybridizable to at least a portion of a target nucleicacid.

In certain embodiments, M₃ is O, CH═CH, OCH₂ or OC(H)(Bx₂). In certainembodiments, M₃ is O.

In certain embodiments, J₄, J₅, J₆ and J₇ are each H. In certainembodiments, J₄ forms a bridge with one of J₅ or J₇.

In certain embodiments, A has one of the formulas:

wherein:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy. In certainembodiments, Q₁ and Q₂ are each H. In certain embodiments, Q₁ and Q₂ areeach, independently, H or halogen. In certain embodiments, Q₁ and Q₂ isH and the other of Q₁ and Q₂ is F, CH₃ or OCH₃.

In certain embodiments, T, has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protectedthiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substitutedC₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S. In certain embodiments, R_(b) is O and R_(a) and R_(c)are each, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃,O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁),O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₀)(R₁₁),OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁₁) orO(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ areeach, independently, H or C₁-C₆ alkyl. In certain embodiments, G ishalogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certainembodiments, G is F, OCH₃ or O(CH₂)₂—OCH₃. In certain embodiments, G isO(CH₂)₂—OCH₃.

In certain embodiments, the 5′-terminal nucleoside has Formula IIe:

In certain embodiments, antisense compounds, including thoseparticularly suitable for ssRNA comprise one or more type of modifiedsugar moieties and/or naturally occurring sugar moieties arranged alongan oligonucleotide or region thereof in a defined pattern or sugarmodification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having uniform sugar modifications. In certain such embodiments,each nucleoside of the region comprises the same RNA-like sugarmodification. In certain embodiments, each nucleoside of the region is a2′-F nucleoside. In certain embodiments, each nucleoside of the regionis a 2′-OMe nucleoside. In certain embodiments, each nucleoside of theregion is a 2′-MOE nucleoside. In certain embodiments, each nucleosideof the region is a cEt nucleoside. In certain embodiments, eachnucleoside of the region is an LNA nucleoside. In certain embodiments,the uniform region constitutes all or essentially all of theoligonucleotide. In certain embodiments, the region constitutes theentire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions ofalternating sugar modifications, wherein the nucleosides alternatebetween nucleotides having a sugar modification of a first type andnucleotides having a sugar modification of a second type. In certainembodiments, nucleosides of both types are RNA-like nucleosides. Incertain embodiments the alternating nucleosides are selected from:2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, thealternating modifications are 2′-F and 2′-OMe. Such regions may becontiguous or may be interrupted by differently modified nucleosides orconjugated nucleosides.

In certain embodiments, the alternating region of alternatingmodifications each consist of a single nucleoside (i.e., the pattern is(AB)_(x)A_(y) wherein A is a nucleoside having a sugar modification of afirst type and B is a nucleoside having a sugar modification of a secondtype; x is 1-20 and y is 0 or 1). In certain embodiments, one or morealternating regions in an alternating motif includes more than a singlenucleoside of a type. For example, oligonucleotides may include one ormore regions of any of the following nucleoside motifs:

AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA;ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of asecond type. In certain embodiments, A and B are each selected from2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternatingmotif also comprise a modified 5′ terminal nucleoside, such as those offormula IIc or IIe.

In certain embodiments, oligonucleotides comprise a region having a2-2-3 motif. Such regions comprises the following motif:

-(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-

wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however,B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certainembodiments, B and C are both 2′-F modified nucleosides. In certainembodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-Fmodified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(AB)_(x)A_(y)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certainembodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from thenucleoside adjacent to it. Thus, if y is 0, then D must be differentlymodified than B and if y is 1, then D must be differently modified thanA. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(A)_(x)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certainembodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selectedfrom: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, Drepresents terminal nucleosides. In certain embodiments, such terminalnucleosides are not designed to hybridize to the target nucleic acid(though one or more might hybridize by chance). In certain embodiments,the nucleobase of each D nucleoside is adenine, regardless of theidentity of the nucleobase at the corresponding position of the targetnucleic acid. In certain embodiments the nucleobase of each D nucleosideis thymine.

In certain embodiments, antisense compounds, including thoseparticularly suited for use as ssRNA comprise modified internucleosidelinkages arranged along the oligonucleotide or region thereof in adefined pattern or modified internucleoside linkage motif. In certainembodiments, oligonucleotides comprise a region having an alternatinginternucleoside linkage motif. In certain embodiments, oligonucleotidescomprise a region of uniformly modified internucleoside linkages. Incertain such embodiments, the oligonucleotide comprises a region that isuniformly linked by phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, eachinternucleoside linkage of the oligonucleotide is selected fromphosphodiester and phosphorothioate. In certain embodiments, eachinternucleoside linkage of the oligonucleotide is selected fromphosphodiester and phosphorothioate and at least one internucleosidelinkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least one 12 consecutive phosphorothioate internucleoside linkages.In certain such embodiments, at least one such block is located at the3′ end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Oligonucleotides having any of the various sugar motifs describedherein, may have any linkage motif. For example, the oligonucleotides,including but not limited to those described above, may have a linkagemotif selected from non-limiting the table below:

5' most linkage Central region 3'-region PS Alternating PO/PS 6 PS PSAlternating PO/PS 7 PS PS Alternating PO/PS 8 PS

ii. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAicompounds (siRNA). In such embodiments, one or both strands may compriseany modification motif described above for ssRNA. In certainembodiments, ssRNA compounds may be unmodified RNA. In certainembodiments, siRNA compounds may comprise unmodified RNA nucleosides,but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein eachstrand comprises a motif defined by the location of one or more modifiedor unmodified nucleosides. In certain embodiments, compositions areprovided comprising a first and a second oligomeric compound that arefully or at least partially hybridized to form a duplex region andfurther comprising a region that is complementary to and hybridizes to anucleic acid target. It is suitable that such a composition comprise afirst oligomeric compound that is an antisense strand having full orpartial complementarity to a nucleic acid target and a second oligomericcompound that is a sense strand having one or more regions ofcomplementarity to and forming at least one duplex region with the firstoligomeric compound.

The compositions of several embodiments modulate gene expression byhybridizing to a nucleic acid target resulting in loss of its normalfunction. In some embodiments, the target nucleic acid is TMPRSS6. Incertain embodiment, the degradation of the targeted TMPRSS6 isfacilitated by an activated RISC complex that is formed withcompositions of the invention.

Several embodiments are directed to double-stranded compositions whereinone of the strands is useful in, for example, influencing thepreferential loading of the opposite strand into the RISC (or cleavage)complex. The compositions are useful for targeting selected nucleic acidmolecules and modulating the expression of one or more genes. In someembodiments, the compositions of the present invention hybridize to aportion of a target RNA resulting in loss of normal function of thetarget RNA.

Certain embodiments are drawn to double-stranded compositions whereinboth the strands comprises a hemimer motif, a fully modified motif, apositionally modified motif or an alternating motif. Each strand of thecompositions of the present invention can be modified to fulfil aparticular role in for example the siRNA pathway. Using a differentmotif in each strand or the same motif with different chemicalmodifications in each strand permits targeting the antisense strand forthe RISC complex while inhibiting the incorporation of the sense strand.Within this model, each strand can be independently modified such thatit is enhanced for its particular role. The antisense strand can bemodified at the 5′-end to enhance its role in one region of the RISCwhile the 3′-end can be modified differentially to enhance its role in adifferent region of the RISC.

The double-stranded oligonucleotide molecules can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The double-stranded oligonucleotide molecules can beassembled from two separate oligonucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (i.e. each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double-stranded structure, for examplewherein the double-stranded region is about 15 to about 30, e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 basepairs; the antisense strand comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense strand comprises nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof(e.g., about 15 to about 25 or more nucleotides of the double-strandedoligonucleotide molecule are complementary to the target nucleic acid ora portion thereof). Alternatively, the double-stranded oligonucleotideis assembled from a single oligonucleotide, where the self-complementarysense and antisense regions of the siRNA are linked by means of anucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The double-stranded oligonucleotide can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprisesseparate sense and antisense sequences or regions, wherein the sense andantisense regions are covalently linked by nucleotide or non-nucleotidelinkers molecules as is known in the art, or are alternatelynon-covalently linked by ionic interactions, hydrogen bonding, van derwaals interactions, hydrophobic interactions, and/or stackinginteractions. In certain embodiments, the double-strandedoligonucleotide comprises nucleotide sequence that is complementary tonucleotide sequence of a target gene. In another embodiment, thedouble-stranded oligonucleotide interacts with nucleotide sequence of atarget gene in a manner that causes inhibition of expression of thetarget gene.

As used herein, double-stranded oligonucleotides need not be limited tothose molecules containing only RNA, but further encompasses chemicallymodified nucleotides and non-nucleotides. In certain embodiments, theshort interfering nucleic acid molecules lack 2′-hydroxy (2′-OH)containing nucleotides. In certain embodiments short interfering nucleicacids optionally do not include any ribonucleotides (e.g., nucleotideshaving a 2′-OH group). Such double-stranded oligonucleotides that do notrequire the presence of ribonucleotides within the molecule to supportRNAi can however have an attached linker or linkers or other attached orassociated groups, moieties, or chains containing one or morenucleotides with 2′-OH groups. Optionally, double-strandedoligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30,40, or 50% of the nucleotide positions. As used herein, the term siRNAis meant to be equivalent to other terms used to describe nucleic acidmolecules that are capable of mediating sequence specific RNAi, forexample short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), short hairpin RNA (shRNA), short interferingoligonucleotide, short interfering nucleic acid, short interferingmodified oligonucleotide, chemically modified siRNA,post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term RNAi is meant to be equivalent toother terms used to describe sequence specific RNA interference, such aspost transcriptional gene silencing, translational inhibition, orepigenetics. For example, double-stranded oligonucleotides can be usedto epigenetically silence genes at both the post-transcriptional leveland the pre-transcriptional level. In a non-limiting example, epigeneticregulation of gene expression by siRNA molecules of the invention canresult from siRNA mediated modification of chromatin structure ormethylation pattern to alter gene expression (see, for example, Verdelet al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science,303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218;and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of severalembodiments provided herein can target TMPRSS6 by a dsRNA-mediated genesilencing or RNAi mechanism, including, e.g., “hairpin” or stem-loopdouble-stranded RNA effector molecules in which a single RNA strand withself-complementary sequences is capable of assuming a double-strandedconformation, or duplex dsRNA effector molecules comprising two separatestrands of RNA. In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, byWO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filedApr. 21, 1999. The dsRNA or dsRNA effector molecule may be a singlemolecule with a region of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In various embodiments, a dsRNA that consistsof a single molecule consists entirely of ribonucleotides or includes aregion of ribonucleotides that is complementary to a region ofdeoxyribonucleotides. Alternatively, the dsRNA may include two differentstrands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely ofribonucleotides, one strand consists entirely of ribonucleotides and onestrand consists entirely of deoxyribonucleotides, or one or both strandscontain a mixture of ribonucleotides and deoxyribonucleotides. Incertain embodiments, the regions of complementarity are at least 70, 80,90, 95, 98, or 100% complementary to each other and to a target nucleicacid sequence. In certain embodiments, the region of the dsRNA that ispresent in a double-stranded conformation includes at least 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or5000 nucleotides or includes all of the nucleotides in a cDNA or othertarget nucleic acid sequence being represented in the dsRNA.

In some embodiments, the dsRNA does not contain any single strandedregions, such as single stranded ends, or the dsRNA is a hairpin. Inother embodiments, the dsRNA has one or more single stranded regions oroverhangs. In certain embodiments, RNA/DNA hybrids include a DNA strandor region that is an antisense strand or region (e.g, has at least 70,80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and anRNA strand or region that is a sense strand or region (e.g, has at least70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and viceversa.

In various embodiments, the RNA/DNA hybrid is made in vitro usingenzymatic or chemical synthetic methods such as those described hereinor those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No.60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strandsynthesized in vitro is complexed with an RNA strand made in vivo or invitro before, after, or concurrent with the transformation of the DNAstrand into the cell. In yet other embodiments, the dsRNA is a singlecircular nucleic acid containing a sense and an antisense region, or thedsRNA includes a circular nucleic acid and either a second circularnucleic acid or a linear nucleic acid (see, for example, WO 00/63364,filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.)Exemplary circular nucleic acids include lariat structures in which thefree 5′ phosphoryl group of a nucleotide becomes linked to the 2′hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modifiednucleotides in which the 2′ position in the sugar contains a halogen(such as fluorine group) or contains an alkoxy group (such as a methoxygroup) which increases the half-life of the dsRNA in vitro or in vivocompared to the corresponding dsRNA in which the corresponding 2′position contains a hydrogen or an hydroxyl group. In yet otherembodiments, the dsRNA includes one or more linkages between adjacentnucleotides other than a naturally-occurring phosphodiester linkage.Examples of such linkages include phosphoramide, phosphorothioate, andphosphorodithioate linkages. The dsRNAs may also be chemically modifiednucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In otherembodiments, the dsRNA contains one or two capped strands, as disclosed,for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No.60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partiallydsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNAmolecules described in U.S. Provisional Application 60/399,998; and U.S.Provisional Application 60/419,532, and PCT/US2003/033466, the teachingof which is hereby incorporated by reference. Any of the dsRNAs may beexpressed in vitro or in vivo using the methods described herein orstandard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to resultin cleavage or the target nucleic acid via RNase H or to result incleavage or sequestration through the RISC pathway. In certain suchembodiments, antisense activity may result from occupancy, wherein thepresence of the hybridized antisense compound disrupts the activity ofthe target nucleic acid. In certain such embodiments, the antisensecompound may be uniformly modified or may comprise a mix ofmodifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode TMPRSS6 include, without limitation,the following: GENBANK Accession NM_153609.2 (incorporated herein as SEQID NO: 1), the complement of GENBANK Accession NT_011520.12 truncatedfrom 16850000 to U.S. Pat. No. 16,897,000 (incorporated herein as SEQ IDNO: 2), GENBANK Accession CR456446.1 (incorporated herein as SEQ ID NO:3), GENBANK Accession No. BC039082.1 (incorporated herein as SEQ ID NO:4), GENBANK Accession No. AY358398.1 (incorporated herein as SEQ ID NO:5), or GENBANK Accession No. DB081153.1 (incorporated herein as SEQ IDNO: 6). In certain embodiments, an antisense compound described hereintargets a nucleic acid sequence encoding TMPRSS6. In certainembodiments, an antisense compound described herein targets the sequenceof any of SEQ ID NOs: 1-6.

It is understood that the sequence set forth in each SEQ ID NO in theexamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor TMPRSS6 can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region may encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the target region.

In certain embodiments, a “target segment” is a smaller, sub-portion ofa target region within a nucleic acid. For example, a target segment canbe the sequence of nucleotides of a target nucleic acid to which one ormore antisense compound is targeted. “5′ target site” refers to the5′-most nucleotide of a target segment. “3′ target site” refers to the3′-most nucleotide of a target segment.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceeding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inTMPRSS6 mRNA levels are indicative of inhibition of TMPRSS6 expression.Reductions in levels of a TMPRSS6 protein are also indicative ofinhibition of TMPRSS6 expression. Further, phenotypic changes areindicative of inhibition of TMPRSS6 expression. For example, an increasein hepcidin expression levels can be indicative of inhibition of TMPRSS6expression. In another example, a decrease in iron accumulation intissues can be indicative of inhibition of TMPRSS6 expression. Inanother example, an increase in the percentage of saturation oftransferrin can be indicative of inhibition of TMPRSS6 expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a TMPRSS6 nucleic acid. The most common mechanismof hybridization involves hydrogen bonding (e.g., Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art (Sambrook andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). Incertain embodiments, the antisense compounds provided herein arespecifically hybridizable with a TMPRSS6 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a TMPRSS6 nucleicacid).

Non-complementary nucleobases between an antisense compound and aTMPRSS6 nucleic acid may be tolerated provided that the antisensecompound remains able to specifically hybridize to the TMPRSS6 nucleicacid. Moreover, an antisense compound may hybridize over one or moresegments of a TMPRSS6 nucleic acid such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least 70%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% complementary to a TMPRSS6 nucleic acid, a target region, targetsegment, or specified portion thereof. Percent complementarity of anantisense compound with a target nucleic acid can be determined usingroutine methods. For example, an antisense compound in which 18 of 20nucleobases of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention.

Percent complementarity of an antisense compound with a region of atarget nucleic acid can be determined routinely using BLAST programs(basic local alignment search tools) and PowerBLAST programs known inthe art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang andMadden, Genome Res., 1997, 7, 649 656). Percent homology, sequenceidentity or complementarity, can be determined by, for example, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), usingdefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, antisense compound may be fully complementary to a TMPRSS6nucleic acid, or a target region, or a target segment or target sequencethereof. As used herein, “fully complementary” means each nucleobase ofan antisense compound is capable of precise base pairing with thecorresponding nucleobases of a target nucleic acid. For example, a 20nucleobase antisense compound is fully complementary to a targetsequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e. linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to, 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no morethan 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a TMPRSS6 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a TMPRSS6 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 12 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least a 9, at least a10, at least an 11, at least a 12, at least a 13, at least a 14, atleast a 15, at least a 16, at least a 17, at least an 18, at least a 19,at least a 20, or more nucleobase portion of a target segment, or arange defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identical to one or more of the antisense compounds or SEQID NOs, or a portion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to a TMPRSS6nucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, at least one of the modified internucleosidelinkages are phosphorothioate linkages. In certain embodiments, eachinternucleoside linkage of an antisense compound is a phosphorothioateinternucleoside linkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprisechemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂are each independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R_(l))—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) andR_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids(BNAs) include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,antisense compounds provided herein include one or more BNA nucleosideswherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof seePCT/US2008/068922 published as WO/2009/006478, published Jan. 8, 2009);4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 publishedas WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (seepublished U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protectinggroup (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008);4′-CH₂—C(H)(CH₃)-2′ (see Zhou et al., J. Org. Chem., 2009, 74, 118-134);and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof see PCT/US2008/066154published as WO 2008/154401, published on Dec. 8, 2008).

Further bicyclic nucleosides have been reported in published literature(see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26)8362-8379; Frieden et al., Nucleic Acids Research. 2003, 21, 6365-6372;Elayadi et al., Curr. Opinion Invens. Drugs. 2001, 2, 558-561; Braaschet al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol.Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4,455-456; Koshkin et al., Tetrahedron, 1998, S4, 3607-3630; Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org.Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,399,845; 7,053,207;7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S.Patent Publication Nos.: US2008-0039618; US2007-0287831; US2004-0171570;U.S. Patent Applications, Ser. Nos. 12/129,154; 61/099,844; 61/097,787;61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574;International applications WO 2007/134181; WO 2005/021570; WO2004/106356; WO 99/14226; and PCT International Applications Nos.:PCT/US2008/068922; PCT/US-2008/066154; and PCT/US2008/064591). Each ofthe foregoing bicyclic nucleosides can be prepared having one or morestereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see PCT international applicationPCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

As used herein, “monocyclic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ carbon atoms of the pentofuranosyl sugarmoiety including without limitation, bridges comprising 1 or from 1 to 4linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, aprotecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H),substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O—or —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently,H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the f-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research. 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′to 2′ bridge wherein such bridges include without limitation,α-L-4′-(CH₂)—O-2′, β-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′,4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′, 4′-CH₂—N(R)-2′,4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, a protecting groupor C₁-C₁₂ alkyl.

In certain embodiment, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(c) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine,5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acidrecognition properties have been described (Koshkin et al., Tetrahedron,1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has alsobeen described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridginggroups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared(Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of oligodeoxyribonucleotide duplexes comprising bicyclicnucleosides for use as substrates for nucleic acid polymerases has alsobeen described (Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel conformationally restricted high-affinityoligonucleotide analog has been described in the art (Singh et al., J.Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H)methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA,and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup, C₁-C₆ alkyl or C₁-C₆ alkoxy.

As used herein, the term “modified tetrahydropyran nucleoside” or“modified THP nucleoside” means a nucleoside having a six-memberedtetrahydropyran “sugar” substituted for the pentofuiranosyl residue innormal nucleosides and can be referred to as a sugar surrogate. ModifiedTHP nucleosides include, but are not limited to, what is referred to inthe art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),manitol nucleic acid (NINA) (see Leumann, Bioorg. Med. Chem., 2002. 10,841-854) or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system asillustrated below.

In certain embodiment, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an oligomeric compound oroligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁,J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following formula:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horváth et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(S-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the cyclohexenyl nucleoside analog to an antisense compound orone of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an antisense compound and the otherof T₃ and T4 is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known inthe art and are suitable as sugar surrogates that can be used to modifynucleosides for incorporation into oligomeric compounds as providedherein (see for example review article: Leumann, Christian J. Bioorg. &Med. Chem., 2002, 10, 841-854). Such ring systems can undergo variousadditional substitutions to further enhance their activity.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O], CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,nucleosides with non-bridging 2′ substituents, such as allyl, amino,azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃”, “2′-O-methyl” or “2′-methoxy”each refers to a nucleoside comprising a sugar comprising an —OCH₃ groupat the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Some representative U.S. patents that teach thepreparation of such modified sugars include without limitation, U.S.:4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633;5,700,920; 5,792,847 and 6,600,032 and International ApplicationPCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 onDec. 22, 2005, and each of which is herein incorporated by reference inits entirety.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications may impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional unmodified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a TMPRSS6nucleic acid comprise one or more modified nucleobases. In certainembodiments, gap-widened antisense oligonucleotides targeted to aTMPRSS6 nucleic acid comprise one or more modified nucleobases. Incertain embodiments, at least one of the modified nucleobases is5-methylcytosine. In certain embodiments, each cytosine is a5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substance for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Antisense compound targeted to a TMPRSS6 nucleic acid can be utilized inpharmaceutical compositions by combining the antisense compound with asuitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes water e.g.,water-for-injection (WFI). A pharmaceutically acceptable diluentincludes saline e.g., phosphate-buffered saline (PBS). Water or salineis a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to a TMPRSS6 nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is water or saline. In certain embodiments, theantisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure herein is also drawn to pharmaceuticallyacceptable salts of antisense compounds, prodrugs, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

Pharmaceutically acceptable salts of the compounds described herein maybe prepared by methods well-known in the art. For a review ofpharmaceutically acceptable salts, see Stahl and Wermuth, Handbook ofPharmaceutical Salts: Properties, Selection and Use (Wiley-VCH,Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides areuseful and are well accepted for therapeutic administration to humans.Accordingly, in one embodiment the compounds described herein are in theform of a sodium salt.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Dosing

In certain embodiments, pharmaceutical compositions are administeredaccording to a dosing regimen (e.g., dose, dose frequency, and duration)wherein the dosing regimen can be selected to achieve a desired effect.The desired effect can be, for example, reduction of TMPRSS6 or theprevention, reduction, amelioration or slowing the progression of adisease, disorder or condition associated with TMPRSS6.

In certain embodiments, the variables of the dosing regimen are adjustedto result in a desired concentration of pharmaceutical composition in asubject. “Concentration of pharmaceutical composition” as used withregard to dose regimen can refer to the compound, oligonucleotide, oractive ingredient of the pharmaceutical composition. For example, incertain embodiments, dose and dose frequency are adjusted to provide atissue concentration or plasma concentration of a pharmaceuticalcomposition at an amount sufficient to achieve a desired effect.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Dosing is also dependent on drug potency andmetabolism. In certain embodiments, dosage is from 0.01 μg to 100 mg perkg of body weight, or within a range of 0.001 mg to 1000 mg dosing, andmay be given once or more daily, weekly, biweekly, monthly, quarterly,semi-annually or yearly, or even once every 2 to 20 years. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 μg to 100 mg per kg of body weight, once or moredaily, to once every 20 years or ranging from 0.001 mg to 1000 mgdosing.

Administration

The compounds or pharmaceutical compositions of the present inventioncan be administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be inhaled (i.e., pulmonary), enteral (i.e.,enteric), parenteral or topical.

In certain embodiments, the compounds and compositions as describedherein are administered parenterally. Parenteral administrationincludes, but is not limited to, intravenous, intra-arterial,subcutaneous, intraperitoneal, intraocular, intramuscular, intracranial,intrathecal, intramedullary, intraventricular or intratumoral injectionor infusion. Parenteral administration also includes intranasaladministration.

In certain embodiments, parenteral administration is by infusion.Infusion can be chronic or continuous or short or intermittent. Incertain embodiments, infused pharmaceutical agents are delivered with apump.

In certain embodiments, parenteral administration is by injection. Theinjection can be delivered with a syringe or a pump. In certainembodiments, the injection is a bolus injection. In certain embodiments,the injection is administered directly to a tissue or organ.

In certain embodiments, formulations for parenteral administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

In certain embodiments, the compounds and compositions as describedherein are administered enterally. Enteric administration includes, butis not limited to, oral, transmucosal, intestinal or rectal (e.g.,suppository, enema). In certain embodiments, formulations for enteraladministration of the compounds or compositions can include, but is notlimited to, pharmaceutical carriers, excipients, powders or granules,microparticulates, nanoparticulates, suspensions or solutions in wateror non-aqueous media, capsules, gel capsules, sachets, tablets orminitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In certain embodiments,enteral formulations are those in which compounds provided herein areadministered in conjunction with one or more penetration enhancers,surfactants and chelators.

In certain embodiments, administration includes pulmonaryadministration. In certain embodiments, pulmonary administrationcomprises delivery of aerosolized oligonucleotide to the lung of asubject by inhalation. Following inhalation by a subject of aerosolizedoligonucleotide, oligonucleotide distributes to cells of both normal andinflamed lung tissue, including alveolar macrophages, eosinophils,epithelium, blood vessel endothelium, and bronchiolar epithelium. Asuitable device for the delivery of a pharmaceutical compositioncomprising a modified oligonucleotide includes, but is not limited to, astandard nebulizer device. Additional suitable devices include drypowder inhalers or metered dose inhalers.

In certain embodiments, pharmaceutical compositions are administered toachieve local rather than systemic exposures. For example, pulmonaryadministration delivers a pharmaceutical composition to the lung, withminimal systemic exposure.

Conjugated Antisense Compounds

In certain embodiments, the oligonucleotides or oligomeric compounds asprovided herein are modified by covalent attachment of one or moreconjugate groups. In general, conjugate groups modify one or moreproperties of the attached oligonucleotide or oligomeric compoundincluding but not limited to pharmacodynamics, pharmacokinetics,stability, binding, absorption, cellular distribution, cellular uptake,charge and clearance. As used herein, “conjugate group” means a radicalgroup comprising a group of atoms that are attached to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties. Conjugate groups areroutinely used in the chemical arts and can include a conjugate linkerthat covalently links the conjugate group to an oligonucleotide oroligomeric compound. In certain embodiments, conjugate groups include acleavable moiety that covalently links the conjugate group to anoligonucleotide or oligomeric compound. In certain embodiments,conjugate groups include a conjugate linker and a cleavable moiety tocovalently link the conjugate group to an oligonucleotide or oligomericcompound. In certain embodiments, a conjugate group has the generalformula:

wherein n is from 1 to about 3, m is 0 when n is 1 or m is 1 when n is 2or 3, j is 1 or 0, k is 1 or 0 and the sum of j and k is at least one.

In certain embodiments, n is 1, j is 1 and k is 0. In certainembodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1,j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. Incertain embodiments, n is 2, j is 0 and k is 1. In certain embodiments,n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and kis 0. In certain embodiments, n is 3, j is 0 and k is 1. In certainembodiments, n is 3, j is 1 and k is 1.

Conjugate groups are shown herein as radicals, providing a bond forforming covalent attachment to an oligomeric compound such as anoligonucleotide. In certain embodiments, the point of attachment on theoligomeric compound is at the 3′-terminal nucleoside or modifiednucleoside. In certain embodiments, the point of attachment on theoligomeric compound is the 3′-oxygen atom of the 3′-hydroxyl group ofthe 3′ terminal nucleoside or modified nucleoside. In certainembodiments, the point of attachment on the oligomeric compound is atthe 5′-terminal nucleoside or modified nucleoside. In certainembodiments the point of attachment on the oligomeric compound is the5′-oxygen atom of the 5′-hydroxyl group of the 5′-terminal nucleoside ormodified nucleoside. In certain embodiments, the point of attachment onthe oligomeric compound is at any reactive site on a nucleoside, amodified nucleoside or an internucleoside linkage.

As used herein, “cleavable moiety” and “cleavable bond” mean a cleavablebond or group of atoms that is capable of being split or cleaved undercertain physiological conditions. In certain embodiments, a cleavablemoiety is a cleavable bond. In certain embodiments, a cleavable moietycomprises a cleavable bond. In certain embodiments, a cleavable moietyis a group of atoms. In certain embodiments, a cleavable moiety isselectively cleaved inside a cell or sub-cellular compartment, such as alysosome. In certain embodiments, a cleavable moiety is selectivelycleaved by endogenous enzymes, such as nucleases. In certainembodiments, a cleavable moiety comprises a group of atoms having one,two, three, four, or more than four cleavable bonds.

In certain embodiments, conjugate groups comprise a cleavable moiety. Incertain such embodiments, the cleavable moiety covalently attaches theoligomeric compound to the conjugate linker. In certain suchembodiments, the cleavable moiety covalently attaches the oligomericcompound to the cell-targeting moiety.

In certain embodiments, a cleavable bond is selected from among: anamide, a polyamide, an ester, an ether, one or both esters of aphosphodiester, a phosphate ester, a carbamate, a di-sulfide, or apeptide. In certain embodiments, a cleavable bond is one of the estersof a phosphodiester. In certain embodiments, a cleavable bond is one orboth esters of a phosphodiester. In certain embodiments, the cleavablemoiety is a phosphodiester linkage between an oligomeric compound andthe remainder of the conjugate group. In certain embodiments, thecleavable moiety comprises a phosphodiester linkage that is locatedbetween an oligomeric compound and the remainder of the conjugate group.In certain embodiments, the cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is attachedto the conjugate linker by either a phosphodiester or a phosphorothioatelinkage. In certain embodiments, the cleavable moiety is attached to theconjugate linker by a phosphodiester linkage. In certain embodiments,the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is a cleavable nucleosideor a modified nucleoside. In certain embodiments, the nucleoside ormodified nucleoside comprises an optionally protected heterocyclic baseselected from a purine, substituted purine, pyrimidine or substitutedpyrimidine. In certain embodiments, the cleavable moiety is a nucleosideselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine.

In certain embodiments, the cleavable moiety is 2′-deoxy nucleoside thatis attached to either the 3′ or 5′-terminal nucleoside of an oligomericcompound by a phosphodiester linkage and covalently attached to theremainder of the conjugate group by a phosphodiester or phosphorothioatelinkage. In certain embodiments, the cleavable moiety is 2′-deoxyadenosine that is attached to either the 3′ or 5′-terminal nucleoside ofan oligomeric compound by a phosphodiester linkage and covalentlyattached to the remainder of the conjugate group by a phosphodiester orphosphorothioate linkage. In certain embodiments, the cleavable moietyis 2′-deoxy adenosine that is attached to the 3′-oxygen atom of the3′-hydroxyl group of the 3′-terminal nucleoside or modified nucleosideby a phosphodiester linkage. In certain embodiments, the cleavablemoiety is 2′-deoxy adenosine that is attached to the 5′-oxygen atom ofthe 5′-hydroxyl group of the 5′-terminal nucleoside or modifiednucleoside by a phosphodiester linkage. In certain embodiments, thecleavable moiety is attached to a 2′-position of a nucleoside ormodified nucleoside of an oligomeric compound.

As used herein, “conjugate linker” in the context of a conjugate groupmeans a portion of a conjugate group comprising any atom or group ofatoms that covalently link the cell-targeting moiety to the oligomericcompound either directly or through the cleavable moiety. In certainembodiments, the conjugate linker comprises groups selected from alkyl,amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether(—S—) and hydroxylamino (—O—N(H)—). In certain embodiments, theconjugate linker comprises groups selected from alkyl, amino, oxo, amideand ether groups. In certain embodiments, the conjugate linker comprisesgroups selected from alkyl and amide groups. In certain embodiments, theconjugate linker comprises groups selected from alkyl and ether groups.In certain embodiments, the conjugate linker comprises at least onephosphorus linking group. In certain embodiments, the conjugate linkercomprises at least one phosphodiester group. In certain embodiments, theconjugate linker includes at least one neutral linking group.

In certain embodiments, the conjugate linker is covalently attached tothe oligomeric compound. In certain embodiments, the conjugate linker iscovalently attached to the oligomeric compound and the branching group.In certain embodiments, the conjugate linker is covalently attached tothe oligomeric compound and a tethered ligand. In certain embodiments,the conjugate linker is covalently attached to the cleavable moiety. Incertain embodiments, the conjugate linker is covalently attached to thecleavable moiety and the branching group. In certain embodiments, theconjugate linker is covalently attached to the cleavable moiety and atethered ligand. In certain embodiments, the conjugate linker includesone or more cleavable bonds. In certain embodiments, the conjugate groupdoes not include a conjugate linker.

As used herein, “branching group” means a group of atoms having at least3 positions that are capable of forming covalent linkages to two or moretether-ligands and the remainder of the conjugate group. In general abranching group provides a plurality of reactive sites for connectingtethered ligands to the oligomeric compound through the conjugate linkerand/or the cleavable moiety. In certain embodiments, the branching groupcomprises groups selected from alkyl, amino, oxo, amide, disulfide,polyethylene glycol, ether, thioether and hydroxylamino groups. Incertain embodiments, the branching group comprises a branched aliphaticgroup comprising groups selected from alkyl, amino, oxo, amide,disulfide, polyethylene glycol, ether, thioether and hydroxylaminogroups. In certain such embodiments, the branched aliphatic groupcomprises groups selected from alkyl, amino, oxo, amide and ethergroups. In certain such embodiments, the branched aliphatic groupcomprises groups selected from alkyl, amino and ether groups. In certainsuch embodiments, the branched aliphatic group comprises groups selectedfrom alkyl and ether groups. In certain embodiments, the branching groupcomprises a mono or polycyclic ring system.

In certain embodiments, the branching group is covalently attached tothe conjugate linker. In certain embodiments, the branching group iscovalently attached to the cleavable moiety. In certain embodiments, thebranching group is covalently attached to the conjugate linker and eachof the tethered ligands. In certain embodiments, the branching groupcomprises one or more cleavable bond. In certain embodiments, theconjugate group does not include a branching group.

In certain embodiments, conjugate groups as provided herein include acell-targeting moiety that has at least one tethered ligand. In certainembodiments, the cell-targeting moiety comprises two tethered ligandscovalently attached to a branching group. In certain embodiments, thecell-targeting moiety comprises three tethered ligands covalentlyattached to a branching group.

As used herein, “tether” means a group of atoms that connect a ligand tothe remainder of the conjugate group. In certain embodiments, eachtether is a linear aliphatic group comprising one or more groupsselected from alkyl, substituted alkyl, ether, thioether, disulfide,amino, oxo, amide, phosphodiester and polyethylene glycol groups in anycombination. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, ether,thioether, disulfide, amino, oxo, amide and polyethylene glycol groupsin any combination. In certain embodiments, each tether is a linearaliphatic group comprising one or more groups selected from alkyl,substituted alkyl, phosphodiester, ether and amino, oxo, amide groups inany combination. In certain embodiments, each tether is a linearaliphatic group comprising one or more groups selected from alkyl, etherand amino, oxo, amide groups in any combination. In certain embodiments,each tether is a linear aliphatic group comprising one or more groupsselected from alkyl, amino and oxo groups in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl and oxo groups in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl and phosphodiester in anycombination. In certain embodiments, each tether comprises at least onephosphorus linking group or neutral linking group.

In certain embodiments, tethers include one or more cleavable bond. Incertain embodiments, each tethered ligand is attached to a branchinggroup. In certain embodiments, each tethered ligand is attached to abranching group through an amide group. In certain embodiments, eachtethered ligand is attached to a branching group through an ether group.In certain embodiments, each tethered ligand is attached to a branchinggroup through a phosphorus linking group or neutral linking group. Incertain embodiments, each tethered ligand is attached to a branchinggroup through a phosphodiester group. In certain embodiments, eachtether is attached to a ligand through either an amide or an ethergroup. In certain embodiments, each tether is attached to a ligandthrough an ether group.

In certain embodiments, each tether comprises from about 8 to about 20atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether comprises from about 10 to about 18atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether comprises about 13 atoms in chainlength.

In certain embodiments, the present disclosure provides ligands whereineach ligand is covalently attached to the remainder of the conjugategroup through a tether. In certain embodiments, each ligand is selectedto have an affinity for at least one type of receptor on a target cell.In certain embodiments, ligands are selected that have an affinity forat least one type of receptor on the surface of a mammalian liver cell.In certain embodiments, ligands are selected that have an affinity forthe hepatic asialoglycoprotein receptor (ASGP-R). In certainembodiments, each ligand is a carbohydrate. In certain embodiments, eachligand is, independently selected from galactose, N-acetylgalactoseamine, mannose, glucose, glucosamone and fucose.

In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc).In certain embodiments, the targeting moiety comprises 1 to 3 ligands.In certain embodiments, the targeting moiety comprises 3 ligands. Incertain embodiments, the targeting moiety comprises 2 ligands. Incertain embodiments, the targeting moiety comprises 1 ligand. In certainembodiments, the targeting moiety comprises 3 N-acetyl galactoseamineligands. In certain embodiments, the targeting moiety comprises 2N-acetyl galactoseamine ligands. In certain embodiments, the targetingmoiety comprises 1 N-acetyl galactoseamine ligand.

In certain embodiments, each ligand is a carbohydrate, carbohydratederivative, modified carbohydrate, multivalent carbohydrate cluster,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain embodiments, each ligand is an amino sugar or a thio sugar.For example, amino sugars may be selected from any number of compoundsknown in the art, for example glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-acetamido-2-deoxy-D-galactopyranose (GalNAc),2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramicacid), 2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom the group consisting of 5-Thio-β-D-glucopyranose, Methyl2,3,4-tri-O-acetyl-1-thio-6-0-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, conjugate groups as provided herein comprise acarbohydrate cluster. As used herein, “carbohydrate cluster” means aportion of a conjugate group wherein two or more carbohydrate residuesare attached to a branching group through tether groups. (see, e.g.,Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to aMultivalent Carbohydrate Cluster for Cellular Targeting,” BioconjugateChemistry, 2003, (14): 18-29, which is incorporated herein by referencein its entirety, or Rensen et al., “Design and Synthesis of NovelN-Acetylgalactosamine-Terminated Glycolipids for Targeting ofLipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem.2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

As used herein, “modified carbohydrate” means any carbohydrate havingone or more chemical modifications relative to naturally occurringcarbohydrates.

As used herein, “carbohydrate derivative” means any compound which maybe synthesized using a carbohydrate as a starting material orintermediate.

As used herein, “carbohydrate” means a naturally occurring carbohydrate,a modified carbohydrate, or a carbohydrate derivative.

In certain embodiments, conjugate groups are provided wherein thecell-targeting moiety has the formula:

In certain embodiments, conjugate groups are provided wherein thecell-targeting moiety has the formula:

In certain embodiments, conjugate groups are provided wherein thecell-targeting moiety has the formula:

In certain embodiments, conjugate groups have the formula:

Representative United States patents, United States patent applicationpublications, and international patent application publications thatteach the preparation of certain of the above noted conjugate groups,conjugated oligomeric compounds such as antisense compounds comprising aconjugate group, tethers, conjugate linkers, branching groups, ligands,cleavable moieties as well as other modifications include withoutlimitation, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182,7,262,177, 7,491,805, 8,106,022, 7,723,509, US 2006/0148740, US2011/0123520, WO 2013/033230 and WO 2012/037254, each of which isincorporated by reference herein in its entirety.

Representative publications that teach the preparation of certain of theabove noted conjugate groups, conjugated oligomeric compounds such asantisense compounds comprising a conjugate group, tethers, conjugatelinkers, branching groups, ligands, cleavable moieties as well as othermodifications include without limitation, BIESSEN et al., “TheCholesterol Derivative of a Triantennary Galactoside with High Affinityfor the Hepatic Asialoglycoprotein Receptor: a Potent CholesterolLowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al.,“Synthesis of Cluster Galactosides with High Affinity for the HepaticAsialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE etal., “New and more efficient multivalent glyco-ligands forasialoglycoprotein receptor of mammalian hepatocytes” Bioorganic &Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determinationof the Upper Size Limit for Uptake and Processing of Ligands by theAsialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J.Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design andSynthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids forTargeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J.Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesisof Novel Amphiphilic Dendritic Galactosides for Selective Targeting ofLiposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem.(1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis oflysine-based cluster galactosides with high affinity for theAsialoglycoprotein Receptor” Tetrahedron. 1997, 53(2). 759-770, each ofwhich is incorporated by reference herein in its entirety.

In certain embodiments, conjugate groups include without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes. Certain conjugate groups have beendescribed previously, for example: cholesterol moiety (Letsinger et al.,Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharanet al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

Some nonlimiting examples of conjugate linkers include pyrrolidine,8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA).Other conjugate linkers include, but are not limited to, substitutedC₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl or substitutedor unsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferredsubstituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof TMPRSS6 nucleic acids can be tested in vitro in a variety of celltypes. Cell types used for such analyses are available from commercialvendors (e.g., American Type Culture Collection, Manassas, Va.; Zen-Bio,Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville,Md.) and cells are cultured according to the vendor's instructions usingcommercially available reagents (e.g., Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells,primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad,Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® inOPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) toachieve the desired concentration of antisense oligonucleotide and aLIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a Cytofectin®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Oligofectamine™ (Invitrogen Life Technologies,Carlsbad, Calif.). Antisense oligonucleotide is mixed withOligofectamine™ in Opti-MEM™-1 reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide with an Oligofectamine™ to oligonucleotide ratio ofapproximately 0.2 to 0.8 μL per 100 nM.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes FuGENE 6 (Roche Diagnostics Corp., Indianapolis,Ind.). Antisense oligomeric compound was mixed with FuGENE 6 in 1 mL ofserum-free RPMI to achieve the desired concentration of oligonucleotidewith a FuGENE 6 to oligomeric compound ratio of 1 to 4 μL of FuGENE 6per 100 nM.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation (Sambrook and Russell inMolecular Cloning. A Laboratory Manual. Third Edition. Cold SpringHarbor laboratory Press, Cold Spring Harbor, N.Y. 2001).

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein (Sambrook and Russell in Molecular Cloning. A Laboratory Manual.Third Edition. Cold Spring Harbor laboratory Press, Cold Spring Harbor,N.Y. 2001). In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art (Sambrook and Russell in Molecular Cloning. A LaboratoryManual. Third Edition. Cold Spring Harbor laboratory Press, Cold SpringHarbor, N.Y. 2001). Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE2000®, Lipofectin or Cytofectin. Antisense oligonucleotidesare used at higher concentrations ranging from 625 to 20,000 nM whentransfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+mRNA.Methods of RNA isolation are well known in the art (Sambrook andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). RNAis prepared using methods well known in the art, for example, using theTRIZOL® Reagent (Invitrogen, Carlsbad, Calif.) according to themanufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a TMPRSS6 nucleic acid can beassayed in a variety of ways known in the art (Sambrook and Russell,Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). For example,target nucleic acid levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or quantitativereal-time PCR. RNA analysis can be performed on total cellular RNA orpoly(A)+mRNA. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Quantitativereal-time PCR can be conveniently accomplished using the commerciallyavailable ABI PRISM® 7600, 7700, or 7900 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN® (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000instrument (PE Applied Biosystems) is used to measure RIBOGREEN®fluorescence.

Probes and primers are designed to hybridize to a TMPRSS6 nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESS®Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of TMPRSS6 nucleic acids can be assessed bymeasuring TMPRSS6 protein levels. Protein levels of TMPRSS6 can beevaluated or quantitated in a variety of ways well known in the art,such as immunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS) (Sambrook and Russell, Molecular Cloning: A LaboratoryManual, 3^(rd) Ed., 2001). Antibodies directed to a target can beidentified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional monoclonal or polyclonal antibody generationmethods well known in the art.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of TMPRSS6 andproduce phenotypic changes, such as, reduced accumulation of iron in thebody. Testing can be performed in normal animals, or in experimentaldisease models. For administration to animals, antisenseoligonucleotides are formulated in a pharmaceutically acceptablediluent, such as sterile water-for-injection or phosphate-bufferedsaline. Administration includes parenteral routes of administration,such as intraperitoneal, intravenous, and subcutaneous. Calculation ofantisense oligonucleotide dosage and dosing frequency depends uponfactors such as route of administration and animal body weight. In oneembodiment, following a period of treatment with antisenseoligonucleotides, RNA is isolated from liver tissue and changes inTMPRSS6 nucleic acid expression are measured. Changes in TMPRSS6 proteinlevels can also be measured. Changes in TMPRSS6 expression can bemeasured by determining the level of hepcidin expression, plasma levelsof iron and percentage saturation of transferrin present in the animal.

Certain Indications

Provided are compositions, compounds and methods for treating anindividual comprising administering to the individual one or morecompositions or compounds described herein. In certain embodiments,compositions, compounds and methods are provided for reducing TMPRSS6expression in the individual. In certain embodiments, compositions,compounds and methods are provided for treating the individual byadministering to the individual a therapeutically effective amount of acomposition or compound comprising an antisense oligonucleotide targetedto a TMPRSS6 nucleic acid. In certain embodiments, the antisensecompound targeted to a TMPRSS6 reduces TMPRSS6. In certain embodiments,the individual in need of TMRPSS6 reduction has, or is at risk for, aniron accumulation disease, disorder or condition. In certainembodiments, compositions, compounds and methods described herein areprovided herein for use in reducing iron levels in an individual.

In certain embodiments, the iron accumulation is the result of a therapyto treat a disease, disorder or condition in the individual. In certainembodiments, the therapy is transfusion therapy. In certain embodiments,multiple transfusions may lead to polycythemia. In further embodiments,multiple blood transfusions are associated with the animal havinganemia. Examples of anemia requiring multiple blood transfusions arehereditary anemia, myelodysplastic syndrome and severe chronichemolysis. Examples of hereditary anemia include, but are not limitedto, sickle cell anemia, thalassemia, Fanconi anemia, Diamond Blackfananemia, Shwachman Diamond syndrome, red cell membrane disorders,glucose-6-phosphate dehydrogenase deficiency, or hereditary hemorrhagictelangiectasia. In certain embodiments, the thalassemia isβ-thalassemia. In certain embodiments, the β-thalassemia isHbE/β-thalassemia, β-thalassemia major, β-thalassemia intermedia orβ-thalassemia minor.

In certain embodiments, the iron accumulation is due to a disease,disorder or condition in the individual. In certain embodiments, thedisease, disorder or condition is hereditary hemochromatosis orthalassemia. In certain embodiments, the thalassemia is non-transfusiondependent thalassemia (NTDT) or β-thalassemia. In certain embodiments,the β-thalassemia is HbE/β-thalassemia, 0-thalassemia major,β-thalassemia intermedia or β-thalassemia minor.

In certain embodiments, the disease, disorder and/or condition isassociated with excess parenteral iron supplement intake or excessdietary iron intake.

Provided herein are compositions, compounds and methods for increasinghepcidin levels, such as mRNA or protein expression levels. In certainembodiments, provided are antisense compounds targeting TMPRSS6 asdescribed herein for use in increasing hepcidin levels, such as mRNA orprotein expression levels.

Provided herein are compositions, compounds and methods for decreasingthe percentage saturation of transferrin in an animal. In certainembodiments, provided are antisense compounds targeting TMPRSS6 asdescribed herein for use in decreasing the percentage saturation oftransferrin in an animal. In certain embodiments, decreasing transferrinsaturation leads to a decrease in iron supply for erythropoiesis. Incertain embodiments, the decrease in erythropoiesis treats, prevents,delays the onset of, ameliorates, and/or reduces polycythemia, orsymptom thereof, in the animal. In certain embodiments, provided areantisense compounds targeting TMPRSS6 as described herein for use intreating, preventing, delaying the onset of, ameliorating, and/orreducing polycythemia, or symptom thereof, in the animal. In certainembodiments, the polycythemia is polycythemia vera. In certainembodiments, treatment with the antisense compound targeting TMPRSS6prevents or delays the polycythemia from progressing into erythroidleukemia.

In certain embodiments, administration of a therapeutically effectiveamount of an antisense compound targeted to a TMPRSS6 nucleic acid in anindividual is accompanied by monitoring of TMPRSS6 levels to determinethe individual's response to the antisense compound. In certainembodiments, administration of a therapeutically effective amount of anantisense compound targeted to a TMPRSS6 nucleic acid in an individualis accompanied by monitoring the levels of hepcidin in the individual.In certain embodiments, administration of a therapeutically effectiveamount of an antisense compound targeted to a TMPRSS6 nucleic acid in anindividual is accompanied by monitoring the levels of iron in theindividual. In certain embodiments, administration of a therapeuticallyeffective amount of an antisense compound targeted to a TMPRSS6 nucleicacid in an individual is accompanied by evaluating the percentagesaturation of transferrin in the individual. An individual's response toadministration of the antisense compound is used by a physician todetermine the amount and duration of therapeutic intervention.

Provided herein are pharmaceutical compositions comprising an antisensecompound targeted to TMPRSS6 for use in the preparation of a medicamentfor treating a patient suffering from, or susceptible to, an ironaccumulation disease, disorder or condition.

In certain embodiments, the methods described herein includeadministering an antisense compound comprising a modifiedoligonucleotide having at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 contiguous nucleobase portion complementary to a TMPRSS6nucleic acid.

Certain Combination Therapies

In certain embodiments, a first agent comprising a composition orcompound provided herein is co-administered with one or more secondaryagents. In certain embodiments, such second agents are designed to treatthe same iron accumulation disease, disorder or condition as the firstagent described herein. In certain embodiments, such second agents aredesigned to treat a different disease, disorder, or condition as thefirst agent described herein. In certain embodiments, such second agentsare designed to treat an undesired side effect of one or morecomposition or compound as described herein. In certain embodiments,such first agents are designed to treat an undesired side effect of asecond agent. In certain embodiments, second agents are co-administeredwith the first agent to treat an undesired effect of the first agent. Incertain embodiments, second agents are co-administered with the firstagent to produce a combinational effect. In certain embodiments, secondagents are co-administered with the first agent to produce a synergisticeffect. In certain embodiments, the co-administration of the first andsecond agents permits use of lower dosages than would be required toachieve a therapeutic or prophylactic effect if the agents wereadministered as independent therapy. In certain embodiments, the dose ofa co-administered second agent is the same as the dose that would beadministered if the second agent was administered alone. In certainembodiments, the dose of a co-administered second agent is lower thanthe dose that would be administered if the second agent was administeredalone. In certain embodiments, the dose of a co-administered secondagent is greater than the dose that would be administered if the secondagent was administered alone.

In certain embodiments, a first agent and one or more second agents areadministered at the same time. In certain embodiments, the first agentand one or more second agents are administered at different times. Incertain embodiments, the second agent is administered prior toadministration of the first agent. In certain embodiments, the secondagent is administered following administration of the first agent. Incertain embodiments, the first agent and one or more second agents areprepared together in a single pharmaceutical formulation. In certainembodiments, the first agent and one or more second agents are preparedseparately.

In certain embodiments, second agents include, but are not limited to,nucleic acid compounds. Such nucleic acid compounds can include a siRNA,a ribozyme or an antisense compound targeting TMPRSS6 or another target.

In certain embodiments, second agents include, but are not limited to,non-antisense compounds such as iron chelators, transferrin, bonemorphogenetic proteins 6 (BMP6), hepcidin agonists, stem cells,antibodies targeting TMPRSS6 or fetal hemoglobin (HbF)-raising agents.In further embodiments, iron chelators are selected from, but notlimited to, FBS0701 (FerroKin), Exjade, Desferal, and Deferiprone. Incertain embodiments, HBF-raising agents include 5-hydroxyl urea, shortchain fatty acid (SCFA) derivatives (e.g., HQK1001), DNAmethyltransferase inhibitors (e.g., decitabine) or histone deacetylase(HDAC) inhibitors (e.g., Zolina, Panobinostat).

In certain embodiments, a second agent includes, but is not limited to,phlebotomy or transfusion therapy. In certain embodiments, the firstagent is administered at the same time as phlebotomy or transfusiontherapy. In certain embodiments, the first agent is administered priorto phlebotomy or transfusion therapy. In certain embodiments, the firstagent is administered following phlebotomy or transfusion therapy. Incertain embodiments, administration of a composition or compoundprovided herein decreases the frequency of phlebotomy or transfusion inan individual. In certain embodiments, administration of a compositionor compound provided herein increases the frequency of phlebotomy ortransfusion in an individual. In certain embodiments, administration ofa composition or compound provided herein decreases the length of timerequired for phlebotomy or transfusion.

Certain Compounds

Preferred antisense compounds with beneficial properties that enhancetheir use as therapeutic treatments in humans are demonstrated in theexamples herein. For brevity, only the studies that contributed to theselection of the preferred antisense compounds are described. Anon-exhaustive summary of the examples is provided below for ease ofreference.

About 2200 antisense compounds with a MOE gapmer motif or a cEtcontaining motif targeting human TMPRSS6 were designed and screened inHep3B cells for their effect on human TMPRSS6 mRNA after administering asingle dose to the cells. Example 1 shows representative single dosescreening data for over 100 potent antisense compounds that wereselected for further studies.

Of the approximately 2200 antisense compounds tested with a single dosein vitro, about 100 antisense compounds were chosen for testing indose-dependent inhibition studies to determine their half maximalinhibitory concentration (IC₅₀) in Hep3B cells (Example 2).

About 77 antisense compounds were further selected, based on theirpotency in dose response and/or single dose studies, for study in CD-1mice to determine tolerability (e.g., plasma chemistry markers, bodyweight and organ weight) of the antisense compound (Examples 3-4) inmice.

Of the approximately 77 antisense compounds tested in CD-1 mice fortolerability, about 48 antisense compounds were chosen for study inSprague-Dawley rats to determine tolerability in rats (Example 5).

Base on the rat tolerability study, about 32 antisense compounds wereselected for in vivo potency testing in human TMPRSS6 transgenic(huTMPRSS6 tg) mice (Example 6).

Antisense compounds identified as potent and tolerable in mice studieswere assessed for cross-reactivity to a rhesus monkey TMPRSS6 genesequence (Example 7). Although the antisense compounds in the studiesdescribed herein were tested in cynomolgus monkeys (Example 11), thecynomolgus monkey TMPRSS6 sequence was not available for comparison tothe sequences of the antisense compounds, therefore the sequences of theantisense compounds were compared to that of the closely related rhesusmonkey. About seven antisense compounds were found to have no mismatcheswith the rhesus TMPRSS6 gene sequence.

Based on the results of the mice potency and tolerability studies, andhomology to the rhesus monkey sequence, the sequences of seven antisensecompounds (585774, 585683, 585775, 630718, 647477, 647449, 647420) fromthe prior studies were selected for further chemical modification tomake them more potent in reducing TMPRSS6 levels. Eight new antisensecompounds with a GalNAc conjugate (702843, 705051, 705052, 705053,706940, 706941, 706942, 706943) were designed based on the sevenoriginal antisense compounds (Example 7).

The eight GalNAc conjugated antisense compounds were tested in mice: fortolerability in CD-1 mice (e.g., body weights, organ weights, livermetabolic markers (e.g., ALT, AST and bilirubin), kidney metabolicmarkers (e.g., BUN and creatinine), histology, hematology parameters(e.g., blood cell counts and hematocrit), and the like were measured(Example 8); and, for potency in human TMPRSS6 transgenic mice (Example9).

The eight GalNAc conjugated antisense compounds were also assessed forviscosity and seven of the eight were found to have a favorableviscosity level while one was found to have a borderline acceptableviscosity level (Example 10).

Based on the favorable profile seen in the mice and in vitro viscositystudies, the eight GalNAc conjugated antisense compounds were furthertested for potency in reducing TMPRSS6, tolerability and for theireffect on iron parameters (e.g., hepcidin levels, serum iron andtransferrin saturation) in cynomolgus monkeys (Example 11). The eightGalNAc conjugated antisense compounds were generally found to be potentand tolerable in cynomolgus monkeys. Antisense compounds 705051, 702843,706942 and 706943 were found to be especially potent in reducingTMRPSS6, serum iron and transferrin saturation.

Accordingly, provided herein are antisense compounds with any one ormore characteristics that are beneficial for their use as a therapeuticagent. In certain embodiments, provided herein are antisense compoundscomprising a modified oligonucleotide as described herein targeted to,or specifically hybridizable with, a region of nucleotides selected fromany of SEQ ID NOs: 1-6.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of their potency in inhibiting TMPRSS6expression. In certain embodiments, the compounds or compositionsinhibit TMPRSS6 by at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90% or at least 95%.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of an in vitro IC₅₀ of less than 20 μM, lessthan 10 μM, less than 8 μM, less than 5 μM, less than 2 μM, less than 1μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6μM, or less than 0.5 μM when tested in human cells, for example, in theHep3B cell line (as described in Example 2).

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of a median effective dose (ED₅₀) of

5 mpk/wk,

4 mpk/wk,

3 mpk/wk,

2 mpk/wk or

1 mpk/wk in vivo. In certain embodiments, preferred antisense compoundshaving an ED₅₀

1 mpk/wk include antisense compounds 702843, 706940, 706942 and 706943as described in Example 8.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of having a viscosity of less than 40 cP, lessthan 35 cP, less than 30 cP, less than 25 cP, less than 20 cP, less than15 cP, or less than 10 cP as described in Example 9. Oligonucleotideshaving a viscosity greater than 40 cP would have less than optimalviscosity.

In certain embodiments, certain antisense compounds as described hereinare highly tolerable, as demonstrated by the in vivo tolerabilitymeasurements described in the examples. In certain embodiments, thecertain antisense compounds as described herein are highly tolerable, asdemonstrated by having an increase in ALT and/or AST value of no morethan 3 fold, 2 fold or 1.5 fold over saline treated animals.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of having one or more of an inhibition potencyof greater than 50%, an ED₅₀≤1 mpk/wk, a viscosity of less than 40 cP,and no more than a 3 fold increase in ALT and/or AST in transgenic mice.

In certain embodiments, ISIS 702843 (SEQ ID NO: 36) is preferred. Thiscompound was found to be a potent inhibitor in TMPRSS6 transgenic miceand a very tolerable antisense compound in CD-1 mice. In mice it hadless than a 3 fold increase in ALT and/or AST levels over saline treatedanimals. It had an acceptable viscosity of about 33 cP and an ED₅₀≤1mpk/wk in huTMPRSS6 transgenic mice. Also, in monkeys, it was among themost potent compounds in inhibiting TMPRSS6.

In certain embodiments, ISIS 705051 (SEQ ID NO: 36) is preferred. Thiscompound was found to be a potent inhibitor in TMPRSS6 transgenic miceand a very tolerable antisense compound in CD-1 mice. In mice it hadless than a 3 fold increase in ALT and/or AST levels over saline treatedanimals. It had an acceptable viscosity of about 23 cP and an ED₅₀≤3mpk/wk in huTMPRSS6 transgenic mice. Also, in monkeys, it was among themost potent compounds in inhibiting TMPRSS6.

In certain embodiments, ISIS 706942 (SEQ ID NO: 77) is preferred. Thiscompound was found to be a potent inhibitor in TMPRSS6 transgenic miceand a very tolerable antisense compound in CD-1 mice. In mice it hadless than a 3 fold increase in ALT and/or AST levels over saline treatedanimals. It had an acceptable viscosity of about 20 cP and an ED₅₀≤1mpk/wk in huTMPRSS6 transgenic mice. Also, in monkeys, it was among themost potent compounds in inhibiting TMPRSS6.

In certain embodiments, ISIS 706943 (SEQ ID NO: 77) is preferred. Thiscompound was found to be a potent inhibitor in TMPRSS6 transgenic miceand a very tolerable antisense compound in CD-1 mice. In huTMPRSS6transgenic mice it had less than a 3 fold increase in ALT and/or ASTlevels over saline treated animals. It had an acceptable viscosity ofabout 19 cP and an ED₅₀≤1 mpk/wk in huTMPRSS6 transgenic mice. Also, inmonkeys, it was among the most potent compounds in inhibiting TMPRSS6.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Antisense Oligonucleotides Targeting Human Type IITransmembrane Serine Protease 6 (TMPRSS6)

Approximately 2200 newly designed chimeric antisense oligonucleotideswere designed as 5-10-5 MOE gapmers or cET containing gapmers.

The 5-10-5 MOE gapmers were designed as oligonucleotides 20 nucleosidesin length, wherein the central gap segment comprises ten2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ direction comprising five nucleosides each. Each nucleosidein the 5′ wing segment and each nucleoside in the 3′ wing segment has a2′-MOE modification. The internucleoside linkages throughout each gapmerare phosphorothioate (P═S) linkages. All cytosine residues throughouteach gapmer are 5-methylcytosines.

The cET containing gapmers were designed with varied deoxy, MOE, and(S)-cEt gapmer motifs. The deoxy, MOE and (S)-cEt oligonucleotides are16 nucleosides in length wherein the nucleosides have either a MOE sugarmodification, an (S)-cEt sugar modification, or a deoxyribose. The‘Chemistry’ column in Table 3 describes the sugar modifications of eacholigonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’indicates deoxyribose; and ‘e’ indicates a MOE modification. Unlessotherwise specified, the internucleoside linkages throughout each gapmerare phosphorothioate (P═S) linkages. All cytosine residues throughouteach gapmer are 5-methylcytosines.

“Start site” indicates the 5′-most nucleoside to which the gapmer istargeted in the human gene sequence. “Stop site” indicates the 3′-mostnucleoside to which the gapmer is targeted human gene sequence. Eachgapmer listed in the Tables below is targeted to either the humanTMPRSS6 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No.NM_153609.2) or the human TMPRSS6 genomic sequence, designated herein asSEQ ID NO: 2 (the complement of GENBANK Accession No. NT_011520.12truncated from nucleotide 16850000 to 16897000). In the tables below,‘n/a’ indicates that the antisense oligonucleotide does not target thatparticular gene sequence with 100% complementarity.

The 2200 chimeric antisense oligonucleotides were tested for theirsingle dose effects on TMRPSS6 mRNA in vitro. Antisense oligonucleotideswere tested at least once in a series of experiments that had similarculture conditions.

A representative result for about 110 potent antisense oligonucleotidesout of the 2200 tested is presented in Tables 1-3 shown below. Thesepotent antisense oligonucleotides were selected for further studies asdescribed below.

Table 1 shows the percent inhibition of TMPRSS6 mRNA by 5-10-5 MOEgapmers. Cultured Hep3B cells at a density of about 20,000 cells perwell were transfected using electroporation with 4,500 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and TMPRSS6 mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3840 (forwardsequence CAAAGCCCAGAAGATGCTCAA, designated herein as SEQ ID NO: 92;reverse sequence GGAATAGACGGAGCTGGAGTTG, designated herein as SEQ ID NO:93; probe sequence ACCAGCACCCGCCTGGGAACTT, designated herein as SEQ IDNO: 94) was used to measure mRNA levels. TMPRSS6 mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of TMPRSS6, relative tountreated control cells.

TABLE 1 Inhibition of TMPRSS6 mRNA by 5-10-5 MOEgapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ IDNO: 1 NO: 1 NO: 2 NO: 2 ISIS Start Stop Start Stop % SEQ ID NO SequenceSite Site Site Site Inhibition NO 585604 CCATCACCTCCGTCCCCCTG 178 1977011 7030 58 7 585606 TCCGCTTCCTCGCCATCACC 190 209 7023 7042 51 8 585608TTTTCTCTTGGAGTCCTCAC 233 252 7066 7085 52 9 585609 GCTTTTCTCTTGGAGTCCTC235 254 7068 7087 79 10 585611 CCGGGCTTTTCTCTTGGAGT 239 258 7072 7091 5811 585626 GGCTTTGGCGGTTTCACTGC 449 468 11948 11967 79 12 585629GAGCATCTTCTGGGCTTTGG 461 480 N/A N/A 80 13 585631 CCTTGAGCATCTTCTGGGCT465 484 N/A N/A 84 14 585649 AGTGCCTGCACCACCTCGGG 616 635 14372 14391 7915 585651 CAGCAGTGCCTGCACCACCT 620 639 14376 14395 70 16 585653TCCTCCACCAGCAGTGCCTG 628 647 14384 14403 49 17 585654AGCTCCTCCACCAGCAGTGC 631 650 14387 14406 64 18 585655CAGCAGCTCCTCCACCAGCA 635 654 14391 14410 66 19 585667GCTGTGCAGGCCCTTCTTCC 1049 1068 24044 24063 52 20 585668GTAGTAGCTGTGCAGGCCCT 1055 1074 24050 24069 61 21 585682ACGGCAAATCATACTTCTGC 1284 1303 26044 26063 60 22 585683GCACGGCAAATCATACTTCT 1286 1305 26046 26065 58 23 585684CCCTGGGTGCACGGCAAATC 1294 1313 26054 26073 58 24 585698CAAACGCAGTTTCTCTCATC 1567 1586 N/A N/A 52 25 585699 TGCAAACGCAGTTTCTCTCA1569 1588 N/A N/A 52 26 585752 GATCACACCTGTGATGCGGG 2504 2523 4426644285 48 27 585757 CTCCTGCCACCACAGGGCCT 2656 2675 44418 44437 70 28585758 ACCTCCTGCCACCACAGGGC 2658 2677 44420 44439 69 29 585761TGCCATCACTGGAGCAGACA 2699 2718 44461 44480 60 30 585762ATCCTCCTGCCATCACTGGA 2706 2725 44468 44487 38 31 585768TCCATTCCCAGATCCCAAGT 2978 2997 44740 44759 64 32 585769CTTCCATTCCCAGATCCCAA 2980 2999 44742 44761 62 33 585770ACCTTCCATTCCCAGATCCC 2982 3001 44744 44763 52 34 585772CAAAGGGCAGCTGAGCTCAC 3154 3173 44916 44935 47 35 585774CTTTATTCCAAAGGGCAGCT 3162 3181 44924 44943 67 36 585775AGCTTTATTCCAAAGGGCAG 3164 3183 44926 44945 68 37 585776AGGCAGCTTTATTCCAAAGG 3168 3187 44930 44949 59 38 585777GATCAGGCAGCTTTATTCCA 3172 3191 44934 44953 65 39 585831AGGAGCGGCCACCGTCCTGT N/A N/A 12340 12359 45 40 12371 12390 12562 12581585834 GGCAGGAGCGGCCACCGTCC N/A N/A 12343 12362 42 41 12374 12393 1256512584 585863 TCCCCCTGAGGCTCTCAGGA N/A N/A 16233 16252 32 42 18737 18756585864 TAAGTCCCCCTGAGGCTCTC N/A N/A 16237 16256 39 43 18741 18760 585906AAGACTGTTCCTTCTCCTTT N/A N/A 27990 28009 44 44 585912CAGCTTGTGCCTGCCCAGAG N/A N/A 29208 29227 45 45 585932AGTCTATCTGGCCACAGTGA N/A N/A 32981 33000 34 46 585937GGTCCTTCTTTGAGCCTCAC N/A N/A 34800 34819 35 47

Table 2 shows the percent inhibition of TMPRSS6 mRNA by additional5-10-5 MOE gapmers. Cultured Hep3B cells at a density of about 20,000cells per well were transfected using electroporation with 5,000 nMantisense oligonucleotide. After a treatment period of approximately 24hours, RNA was isolated from the cells and TMPRSS6 mRNA levels weremeasured by quantitative real-time PCR. Human primer probe set RTS3840was used to measure mRNA levels. TMPRSS6 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of TMPRSS6, relative to untreatedcontrol cells.

TABLE 2 Inhibition of TMPRSS6 mRNA by 5-10-5 MOEgapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ IDNO: 1 NO: 1 NO: 2 NO: 2 ISIS Start Stop Start Stop % SEQ ID NO SequenceSite Site Site Site Inhibition NO 591466 CCTCAGGTCACCACTTGCTG 2533 255244295 44314 63 48 591491 GCCACCTCCTGCCACCACAG 2661 2680 44423 44442 7249 591492 ATGCCACCTCCTGCCACCAC 2663 2682 44425 44444 59 50 591514CTCCATCCTCCTGCCATCAC 2710 2729 44472 44491 59 51 591536GCAGCTGAGCTCACCTCCCA 3148 3167 44910 44929 68 52 591537GGCAGCTGAGCTCACCTCCC 3149 3168 44911 44930 75 53 591549GGCAGCTTTATTCCAAAGGG 3167 3186 44929 44948 69 54 591550CAGGCAGCTTTATTCCAAAG 3169 3188 44931 44950 76 55 591552ATCAGGCAGCTTTATTCCAA 3171 3190 44933 44952 66 56 591578CCACTGGCCCTGGGTGCACG 1301 1320 26061 26080 65 57 591579TCCACTGGCCCTGGGTGCAC 1302 1321 26062 26081 68 58

Table 3 shows the percent inhibition of TMPRSS6 mRNA by cEt containinggapmers from a series of experiments. Cultured Hep3B cells at a densityof about 20,000 cells per well were transfected using electroporationwith 2,000 nM antisense oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and TMPRSS6 mRNAlevels were measured by quantitative real-time PCR. Human primer probeset RTS3840 was used to measure mRNA levels. TMPRSS6 mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of TMPRSS6, relative tountreated control cells.

TABLE 3 Inhibition of TMPRSS6 mRNA by cEt containinggapmers targeting SEQ ID NO: 1 and/or 2 SEQ ID SEQ ID SEQ ID SEQ IDNO: 1 NO: 1 NO: 2 NO: 2 ISIS Start Stop Start Stop % SEQ ID NO SequenceSite Site Site Site Chemistry Inhibition NO 615840 CTTTTGGCTTACAGTG 30573072 44819 44834 ekk-d10-kke 59 59 615884 GCTGAGCTCACCTCCC 3149 316444911 44926 ekk-d10-kke 70 60 615898 TATTCCAAAGGGCAGC 3163 3178 4492544940 ekk-d10-kke 69 61 615901 CTTTATTCCAAAGGGC 3166 3181 44928 44943ekk-d10-kke 68 62 615903 AGCTTTATTCCAAAGG 3168 3183 44930 44945ekk-d10-kke 70 63 615909 TCAGGCAGCTTTATTC 3174 3189 44936 44951ekk-d10-kke 69 64 615910 ATCAGGCAGCTTTATT 3175 3190 44937 44952ekk-d10-kke 69 65 615911 GATCAGGCAGCTTTAT 3176 3191 44938 44953ekk-d10-kke 69 66 630497 ATTCCAAAGGGCAGCT 3162 3177 44924 44939kkk-d10-kkk 80 67 630689 CTTACAGTGGCAGCAG 3050 3065 44812 44827kkk-d10-kkk 71 68 630692 TGGCTTACAGTGGCAG 3053 3068 44815 44830kkk-d10-kkk 75 69 630693 TTGGCTTACAGTGGCA 3054 3069 44816 44831kkk-d10-kkk 75 70 630696 CTTTTGGCTTACAGTG 3057 3072 44819 44834kkk-d10-kkk 66 59 630716 CTTTATTCCAAAGGGC 3166 3181 44928 44943kkk-d10-kkk 63 62 630717 GCTTTATTCCAAAGGG 3167 3182 44929 44944kkk-d10-kkk 81 71 630718 AGCTTTATTCCAAAGG 3168 3183 44930 44945kkk-d10-kkk 84 63 630719 CAGGCAGCTTTATTCC 3173 3188 44935 44950kkk-d10-kkk 80 72 630722 GATCAGGCAGCTTTAT 3176 3191 44938 44953kkk-d10-kkk 72 66 630725 TTTGATCAGGCAGCTT 3179 3194 N/A N/A kkk-d10-kkk61 73 630726 TTTTGATCAGGCAGCT 3180 3195 N/A N/A kkk-d10-kkk 72 74 630727TTTTTGATCAGGCAGC 3181 3196 N/A N/A kkk-d10-kkk 73 75 630794ACATCAGGGACGAGAC 2686 2701 44448 44463 kk-d8-kekeke 72 76 647393TTATTCCAAAGGGCAG 3164 3179 44926 44941 kkk-d10-kkk 78 83 647394TTTATTCCAAAGGGCA 3165 3180 44927 44942 kkk-d10-kkk 77 84 647395CAGCTTTATTCCAAAG 3169 3184 44931 44946 kkk-d10-kkk 86 77 647396GCAGCTTTATTCCAAA 3170 3185 44932 44947 kkk-d10-kkk 86 78 647397GGCAGCTTTATTCCAA 3171 3186 44933 44948 kkk-d10-kkk 85 82 647398AGGCAGCTTTATTCCA 3172 3187 44934 44949 kkk-d10-kkk 82 79 647404GGCAGCTGAGCTCACC 3153 3168 44915 44930 kek-d9-eekk 76 85 647414TATTCCAAAGGGCAGC 3163 3178 44925 44940 kek-d9-eekk 86 61 647419AGCTTTATTCCAAAGG 3168 3183 44930 44945 kek-d9-eekk 87 63 647420CAGCTTTATTCCAAAG 3169 3184 44931 44946 kek-d9-eekk 83 77 647421GCAGCTTTATTCCAAA 3170 3185 44932 44947 kek-d9-eekk 83 78 647423AGGCAGCTTTATTCCA 3172 3187 44934 44949 kek-d9-eekk 84 79 647424CAGGCAGCTTTATTCC 3173 3188 44935 44950 kek-d9-eekk 78 72 647426ATCAGGCAGCTTTATT 3175 3190 44937 44952 kek-d9-eekk 81 65 647428TGATCAGGCAGCTTTA 3177 3192 N/A N/A kek-d9-eekk 76 80 647429TTGATCAGGCAGCTTT 3178 3193 N/A N/A kek-d9-eekk 78 81 647442ATTCCAAAGGGCAGCT 3162 3177 44924 44939 kk-d9-eeekk 81 67 647446CTTTATTCCAAAGGGC 3166 3181 44928 44943 kk-d9-eeekk 79 62 647447GCTTTATTCCAAAGGG 3167 3182 44929 44944 kk-d9-eeekk 87 71 647448AGCTTTATTCCAAAGG 3168 3183 44930 44945 kk-d9-eeekk 86 63 647449CAGCTTTATTCCAAAG 3169 3184 44931 44946 kk-d9-eeekk 89 77 647450GCAGCTTTATTCCAAA 3170 3185 44932 44947 kk-d9-eeekk 88 78 647451GGCAGCTTTATTCCAA 3171 3186 44933 44948 kk-d9-eeekk 88 82 647453CAGGCAGCTTTATTCC 3173 3188 44935 44950 kk-d9-eeekk 77 72 647454TCAGGCAGCTTTATTC 3174 3189 44936 44951 kk-d9-eeekk 82 64 647457TGATCAGGCAGCTTTA 3177 3192 N/A N/A kk-d9-eeekk 78 80 647475CTTTATTCCAAAGGGC 3166 3181 44928 44943 kk-d8-eeeekk 77 62 647476GCTTTATTCCAAAGGG 3167 3182 44929 44944 kk-d8-eeeekk 83 71 647477AGCTTTATTCCAAAGG 3168 3183 44930 44945 kk-d8-eeeekk 84 63 647478CAGCTTTATTCCAAAG 3169 3184 44931 44946 kk-d8-eeeekk 79 77 647482CAGGCAGCTTTATTCC 3173 3188 44935 44950 kk-d8-eeeekk 76 72 647506AGCTTTATTCCAAAGG 3168 3183 44930 44945 k-d9-kekeke 89 63 647508GCAGCTTTATTCCAAA 3170 3185 44932 44947 k-d9-kekeke 77 78 647514GATCAGGCAGCTTTAT 3176 3191 44938 44953 k-d9-kekeke 78 66 647531CAGCTTTATTCCAAAG 3169 3184 44931 44946 kk-d8-kekeke 88 77 647532GCAGCTTTATTCCAAA 3170 3185 44932 44947 kk-d8-kekeke 77 78

Example 2: Dose Response of Antisense Oligonucleotides Targeting HumanTMPRSS6 in Hep3B Cells

About 100 antisense oligonucleotides selected from the about 2200antisense oligonucleotides tested in single dose experiments describedin Example 1 were also tested at various doses in Hep3B cells in studiesof in vitro inhibition of human TMPRSS6 mRNA.

For the experiment in Table 4, below, cells were plated at a density of12,000 cells per well and transfected using electroporation with 0.15μM, 0.44 μM, 1.33 μM, 4.00 μM and 12.00 μM concentrations of antisenseoligonucleotide. After the treatment period of approximately 16 hours,RNA was isolated from the cells and TMPRSS6 mRNA levels were measured byquantitative real-time PMR Human primer probe set RTS3840 was used tomeasure mRNA levels. TMPRSS6 mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of TMPRSS6, relative to untreated control cells. “0”indicate that the antisense oligonucleotide did not reduce TMPRSS6 mRNAlevels.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. TMPRSS6 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 4 Dose response assay with 5-10-5 MOE gapmers 0.15 0.44 1.33 4.0012.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 585604 0 0 17 36 63 7 585606 0 00 0 35 >12 585608 0 13 6 8 50 >12 585609 0 10 24 44 68 5 585611 0 0 9 3367 8 585626 3 21 27 55 82 3 585629 37 45 56 71 83 1 585631 29 56 63 7084 1 585649 0 9 35 46 74 4 585651 0 18 1 39 75 6 585653 10 15 18 42 63 7585654 0 0 25 33 65 8 585655 0 12 15 34 65 8 585667 0 0 2 30 52 >12585668 11 6 0 43 70 8 585682 0 0 0 30 63 11 585683 1 9 19 39 77 5 5856846 1 13 21 57 >12 585698 13 11 37 39 78 4 585699 0 8 25 25 65 8 585752 012 37 34 69 5 585757 0 7 16 53 79 4 585758 6 0 25 49 71 5 585761 2 12 1339 66 7 585762 2 15 26 44 75 4 585768 4 0 20 52 76 4 585769 0 0 0 42 707 585770 12 12 42 50 68 3 585772 12 12 23 34 56 12 585774 15 28 58 68 841 585775 0 7 28 60 82 3 585776 36 24 56 69 86 1 585777 15 39 63 76 88 1585831 0 8 3 19 31 >12 585834 0 10 3 6 32 >12 585863 7 7 3 0 51 >12585864 5 9 19 31 34 >12 585906 13 2 16 11 29 >12 585912 20 0 30 3332 >12 585932 15 11 25 4 37 >12 585937 20 33 30 30 43 >12 591466 0 14 2639 71 5 591491 0 11 23 45 68 5 591492 0 0 22 27 64 9 591514 0 0 1 41 756 591536 13 22 34 64 81 2 591537 17 44 57 81 88 1 591549 21 26 51 72 871 591550 19 34 65 76 89 1 591552 23 49 65 86 90 1 591578 0 17 28 45 55 7591579 3 13 47 40 58 6

For the experiment in Table 5, below, cells were plated at a density of5,000 cells per well and transfected using electroporation with 0.19 μM,0.56 μM, 1.67 μM and 5.0 μM concentrations of antisense oligonucleotide.After the treatment period of approximately 16 hours, RNA was isolatedfrom the cells and TMPRSS6 mRNA levels were measured by quantitativereal-time PCR. Human primer probe set RTS3840 was again used to measuremRNA levels. TMPRSS6 mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of TMPRSS6, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. TMPRSS6 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 5 Dose response assay with cEt containing oligonucleotides 0.190.56 1.67 5.00 IC₅₀ ISIS No μM μM μM μM μM 630497 28 49 69 86 0.6 64739328 42 69 84 0.7 647394 43 59 67 83 0.3 647395 11 41 67 83 0.9 647396 2547 73 79 0.7 647397 27 42 70 83 0.7 647398 27 49 61 84 0.7 647404 23 4763 79 0.8 647414 38 52 72 87 0.4 647419 45 60 74 84 0.3 647420 28 52 6982 0.6 647421 23 47 68 85 0.7 647423 23 50 74 81 0.7 647424 20 48 72 830.7 647426 26 37 67 76 0.9 647428 25 33 61 83 0.9 647429 20 32 59 83 1647442 32 51 66 78 0.6 647446 32 48 73 81 0.6 647447 29 52 70 81 0.6647448 30 56 72 79 0.5 647449 31 45 71 83 0.6 647450 32 54 70 82 0.5647451 40 62 74 83 0.3 647453 28 52 68 84 0.6 647454 32 45 62 84 0.7647457 28 46 69 80 0.7 647475 9 52 63 77 1 647476 43 59 70 79 0.3 64747748 62 77 83 0.2 647478 16 41 68 82 0.9 647482 14 37 73 79 0.9 647506 3760 75 83 0.4 647508 21 39 52 79 1.1 647514 32 42 63 81 0.7 647531 25 5373 80 0.6 647532 26 49 61 82 0.7

For the experiment in Table 6, below, cells were plated at a density of20,000 cells per well and transfected using electroporation with 0.22μM, 0.67 μM, 2.00 μM and 6.0 μM concentrations of antisenseoligonucleotide. After the treatment period of approximately 16 hours,RNA was isolated from the cells and TMPRSS6 mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3840 was used tomeasure mRNA levels. TMPRSS6 mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of TMPRSS6, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. TMPRSS6 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 6 Dose response assay with cEt containing oligonucleotides 0.220.67 2.00 6.00 IC₅₀ ISIS No μM μM μM μM μM 630497 34 54 81 89 0.5 63068943 61 77 87 0.3 630692 54 64 85 95 0.2 630693 42 66 75 86 0.3 630696 2037 66 82 1.1 630717 48 73 84 83 0.1 630718 49 81 88 89 0.1 630719 42 6983 95 0.3 630722 40 56 70 90 0.4 630726 24 45 64 82 0.9 630727 36 57 7382 0.5 630794 25 46 71 84 0.8

Example 3: Tolerability of 5-10-5 MOE Gapmers Targeting Human TMPRSS6 inCD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose mice model,frequently utilized for safety and efficacy testing. The mice weretreated with about 26 ISIS 5-10-5 MOE gapmer antisense oligonucleotidesselected from the tables above and evaluated for changes in the levelsof various plasma chemistry markers.

Treatment

Groups of six week old male CD1 mice were injected subcutaneously twicea week for six weeks with 50 mg/kg of ISIS oligonucleotides (100mg/kg/week dose). One group of male CD1 mice was injected subcutaneouslytwice a week for 6 weeks with PBS. Mice were euthanized 48 hours afterthe last dose, and organs and plasma were harvested for furtheranalysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases (ALT and AST), total bilirubin(Tbil), albumin (Alb), creatinine (Creat), and BUN were measured usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). The results are presented in Table 7. ISISoligonucleotides that caused changes in the levels of any of the liveror kidney function markers outside the expected range for antisenseoligonucleotides were excluded in further studies.

TABLE 7 Plasma chemistry markers in CD1 mice at week six ALT AST BUNCreat Tbil Alb ISIS No. (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) (g/dL) PBS24 51 27 0.17 0.17 2.9 585626 167 155 30 0.18 0.15 2.9 585649 263 157 280.17 0.15 3.0 585653 147 89 28 0.18 0.39 3.4 585654 778 300 26 0.15 0.173.0 585655 1709 1353 29 0.16 0.35 3.0 585683 45 63 31 0.18 0.20 3.0585698 53 73 34 0.21 0.19 3.0 585752 90 99 29 0.16 0.17 2.9 585757 246180 30 0.16 0.15 2.8 585758 212 305 28 0.18 0.28 2.9 585761 659 439 280.16 0.43 2.7 585762 597 551 27 0.17 0.64 3.0 585768 483 387 26 0.180.19 2.7 585774 109 126 31 0.16 0.14 2.6 585775 60 70 28 0.17 0.15 2.9585776 654 388 27 0.17 0.13 2.9 585777 159 200 24 0.16 0.17 2.7 59146646 53 27 0.15 0.12 3.0 591491 761 729 28 0.18 0.25 3.2 591514 230 215 330.15 0.14 2.5 591536 540 416 26 0.16 0.13 3.0 591537 552 346 27 0.170.16 3.0 591549 708 488 30 0.14 0.14 2.7 591550 294 225 31 0.17 0.12 2.9591552 1098 680 24 0.17 0.17 3.0 591579 135 85 25 0.16 0.12 2.8

Body and Organ Weights

Body weights of all the groups of mice were measured at the start of theexperiment, and every week until the end of the study. Liver, spleen andkidney weights were also measured at the end of the study, and thechange in body weight and organ weights relative to the PBS controlgroup at baseline are presented in Table 8. ISIS oligonucleotides thatcaused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 8 Body weight and relative organ weights of CD1 mice (in grams) atweek six BW Relative Relative Relative change liver kidney spleen ISISNo. (g) weight (g) weight (g) weight (g) PBS 1.4 1.0 1.0 1.0 585626 1.41.2 0.9 1.1 585649 1.3 1.2 1.0 1.1 585653 1.4 1.1 1.0 0.9 585654 1.2 1.21.0 1.1 585655 1.3 1.4 1.0 1.3 585683 1.4 1.0 0.9 1.1 585698 1.5 1.2 1.01.4 585752 1.3 1.1 1.0 1.3 585757 1.4 1.5 1.0 1.1 585758 1.4 1.4 0.9 1.0585761 1.1 1.4 1.0 1.3 585762 1.2 2.1 1.0 0.8 585768 1.5 1.1 1.1 1.3585774 1.5 1.1 1.0 1.1 585775 1.5 0.9 1.0 1.2 585776 1.4 1.3 1.1 1.5585777 1.4 1.2 1.1 1.5 591466 1.5 1.0 1.0 1.0 591491 1.3 1.2 1.0 1.1591514 1.4 1.1 0.9 1.5 591536 1.4 1.3 1.0 1.1 591537 1.3 1.3 0.9 1.3591549 1.4 1.2 1.0 1.5 591550 1.4 1.1 0.9 1.5 591552 1.4 1.5 1.1 1.5591579 1.5 1.0 0.9 1.1

From these tolerability studies, it was observed that most of the 5-10-5MOE gapmer antisense oligonucleotides were well-tolerated after sixweeks of dosing.

Example 4: Tolerability of cEt Containing Oligonucleotides TargetingHuman TMPRSS6 in CD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose mice model,frequently utilized for safety and efficacy testing. The mice weretreated with about 51 cEt containing antisense oligonucleotides selectedfrom the tables described above, and evaluated for changes in the levelsof various plasma chemistry markers.

Treatment

Groups of five-to six-week-old male CD1 mice (n=4 per treatment group)were injected subcutaneously twice a week for six weeks with 25 mg/kg ofISIS oligonucleotides (50 mg/kg/week dose). One group of male CD 1 micewas injected subcutaneously twice a week for 6 weeks with PBS. Mice wereeuthanized 48 hours after the last dose, and organs and plasma wereharvested for further analysis. Liver, kidney and spleen were collectedfor histology, and plasma was collected to measure levels of certainplasma chemistry markers.

The oligonucleotides were split into two test groups with the sameconditions and the results are presented to in the tables below.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilinibin, albumin,creatinine, and BUN were measured using an automated clinical chemistryanalyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results arepresented in Tables 9-10. ISIS oligonucleotides causing changes in thelevels of any of the liver or kidney function markers outside theexpected range for antisense oligonucleotides were excluded from furtherstudies.

TABLE 9 Plasma chemistry markers in CD1 mice at week six ALT AST BUNCreat Tbil Alb ISIS No. (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) (g/dL) PBS55 53 24 0.1 0.2 2.7 615840 752 636 26 0.15 0.23 2.5 615884 1039 664 250.17 0.17 2.8 615898 754 420 25 0.17 0.14 2.5 615901 118 120 22 0.110.18 2.5 615903 33 46 22 0.12 0.18 2.5 615909 2042 2464 49 0.16 1.19 2.7615910 978 1058 22 0.15 1.24 2.4 615911 474 366 23 0.14 0.34 2.4 6306961117 853 26 0.15 0.21 2.3 630716 41 67 25 0.13 0.14 2.4 630717 1005 48323 0.13 0.19 2.3 630718 57 86 25 0.13 0.13 2.4 630722 207 168 21 0.130.16 2.2 630725 1729 897 20 0.12 0.15 2.2 630726 1330 774 22 0.10 0.102.1 630727 614 653 23 0.10 0.13 1.6 630794 39 78 24 0.12 0.16 2.6

TABLE 10 Plasma chemistry markers in CD1 mice at week six ALT AST BUNCreat Tbil Alb ISIS No. (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) (g/dL) PBS31.3 54.8 32.3 0.14 0.19 3.0 630497 429.0 297.5 31.0 0.18 0.11 2.8630689 2088.3 1306.0 34.7 0.10 0.22 2.2 630692 1634.8 1402.5 30.9 0.160.25 3.4 630693 1247.5 1193.8 33.6 0.19 0.68 2.8 630719 2553.0 2594.728.6 0.12 2.55 3.8 647414 718.5 444.0 32.7 0.13 0.12 3.0 647419 39.366.5 27.0 0.13 0.15 2.9 647420 90.3 100.8 30.8 0.13 0.19 3.1 647421613.3 607.3 15.5 0.09 1.61 2.6 647423 1290.3 807.5 29.8 0.28 0.30 3.7647424 1451.0 1198.3 25.2 0.16 0.37 3.7 647426 548.5 393.0 23.7 0.120.16 2.7 647428 2658.8 2232.8 24.8 0.21 0.52 3.0 647429 1306.3 725.323.2 0.12 0.21 2.8 647442 564.8 371.5 29.7 0.08 0.13 3.0 647446 69.091.3 27.6 0.10 0.14 2.9 647447 61.5 76.3 27.2 0.11 0.13 2.8 647448 100.8110.5 24.4 0.10 0.14 2.9 647449 61.3 88.0 27.7 0.10 0.13 3.1 6474501850.8 1512.0 18.3 0.09 0.47 2.9 647451 1376.3 588.3 26.0 0.15 0.29 3.7647453 1774.3 1674.5 28.8 0.16 1.24 3.7 647454 324.3 409.3 27.0 0.110.15 2.7 647457 1609.0 1194.8 25.6 0.12 0.21 2.6 647475 40.0 80.5 25.10.10 0.12 2.6 647476 62.0 81.0 26.1 0.11 0.14 2.8 647477 74.8 94.0 26.50.11 0.15 2.9 647478 62.0 88.0 28.2 0.11 0.13 3.1 647482 959.8 975.825.8 0.11 0.19 2.9 647506 36.3 65.3 25.8 0.10 0.14 2.9 647508 49.8 93.326.3 0.11 0.14 3.1 647514 276.0 221.8 28.3 0.11 0.17 2.9 647531 248.5175.0 28.7 0.11 0.16 3.2 647532 156.8 180.0 21.3 0.09 0.10 3.0

Body and Organ Weights

Body weights of all the groups of mice were measured at the start of theexperiment, and every week until the end of the study. Liver, spleen andkidney weights were also measured at the end of the study, and thechange in body weight and organ weights relative to the PBS controlgroup at baseline are presented in Tables 11-12. ISIS oligonucleotidesthat caused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 11 Body weight and relative organ weights of CD1 mice (in grams)at week six BW Relative Relative Relative change liver kidney spleenISIS No. (g) weight (g) weight (g) weight (g) PBS 1.5 1 1 1 615840 1.21.1 1.0 0.8 615884 1.4 1.5 1.1 1.2 615898 1.5 1.3 1.1 1.4 615901 1.5 1.31.1 2.0 615903 1.4 1.1 1.1 1.2 615909 0.8 1.6 1.2 0.7 615910 1.2 1.9 1.02.3 615911 1.5 1.4 1.1 1.6 630696 1.1 1.2 0.9 1.2 630716 1.4 1.2 1.2 1.2630717 1.2 1.4 1.0 1.7 630718 1.4 1.2 1.1 1.4 630722 1.6 1.2 1.1 1.6630725 1.3 1.2 1.1 1.8 630726 1.4 1.1 1.2 1.9 630727 1.3 1.2 1.2 3.5630794 1.4 1.0 1.1 1.1

TABLE 12 Body weight and relative organ weights of CD1 mice (in grams)at week six BW Relative Relative Relative change liver kidney spleenISIS No. (g) weight (g) weight (g) weight (g) PBS 1.5 1 1 1 630497 1.31.2 1.0 1.1 630689 1.6 1.3 1.0 1.4 630692 1.5 1.9 0.9 1.2 630693 1.2 1.30.8 0.9 630719 0.8 1.4 1.1 0.4 647414 1.4 1.2 1.1 1.0 647419 1.5 1.0 1.11.2 647420 1.4 1.1 1.0 1.4 647421 1.2 1.1 1.1 1.3 647423 1.4 1.7 1.1 1.3647424 1.1 1.8 1.2 0.6 647426 1.4 1.5 1.1 1.8 647428 1.3 1.4 1.1 1.9647429 1.4 1.2 1.0 1.6 647442 1.3 1.1 1.1 1.1 647446 1.4 1.2 1.2 1.4647447 1.5 1.3 1.2 1.4 647448 1.5 1.1 1.1 1.5 647449 1.5 1.1 1.1 1.6647450 1.4 1.3 1.1 1.9 647451 1.4 1.6 1.0 1.8 647453 1.2 1.8 1.4 1.5647454 1.5 1.6 1.0 2.2 647457 1.4 1.3 1.0 1.8 647475 1.4 1.2 1.1 1.5647476 1.5 1.1 1.2 1.8 647477 1.5 1.2 1.0 1.5 647478 1.6 1.1 1.0 1.2647482 1.4 1.7 1.2 1.5 647506 1.5 1.1 1.0 1.2 647508 1.6 1.0 1.0 1.2647514 1.5 1.0 1.0 1.5 647531 1.4 1.0 1.0 1.4 647532 1.5 1.3 1.1 1.4

Example 5: Tolerability of Oligonucleotides Targeting Human TMPRSS6 inSprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety andefficacy evaluations. The rats were treated with about 48 antisenseoligonucleotides, found potent in vitro and tolerable in mice from thestudies described in the Examples above, and evaluated for changes inthe levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats (roughly eight weeks old) were maintained on a12-hour light/dark cycle and fed ad libitum with Purina normal rat chow,diet 5001. Groups of four Sprague-Dawley rats each were injectedsubcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmer, or50 mg/kg of cEt containing antisense oligonucleotides. One to two daysafter the final dose, urine protein/creatinine (P/C) ratio was assayedand blood was drawn 3 days after the last dose for hematologicassessments described below. Three days after the last dose, rats wereeuthanized and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases (alanine transaminase (ALT) andaspartate transaminase (AST), total bilirubin (Tbil), albumin (Alb),creatinine (Creat), and BUN were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in Table 13. ISIS oligonucleotides that caused changes inthe levels of any of the liver or kidney function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 13 Plasma chemistry markers in Sprague-Dawley rats ALT AST BUNCreat Tbil Alb ISIS No. (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (g/dL) PBS60 92 18 0.3 0.1 3.7 585626 66 139 25 0.4 0.1 3.2 585653 92 154 26 0.40.1 3.9 585683 73 109 19 0.4 0.1 3.3 585698 66 104 22 0.4 0.1 3.4 58575264 145 21 0.4 0.1 3.0 585758 113 669 21 0.3 0.2 2.8 585774 125 220 250.4 0.2 3.2 585775 66 117 24 0.4 0.1 3.2 585777 302 321 25 0.4 0.2 3.4591466 368 444 22 0.4 0.2 3.1 591514 91 218 22 0.3 0.2 3.3 591579 484655 20 0.4 0.2 3.8 614954 146 132 26 0.1 0.2 2.8 615895 291 383 26 0.40.2 3.4 615897 1946 1467 26 0.5 0.2 4.0 615899 70 113 25 0.4 0.1 3.4615900 93 131 26 0.4 0.1 3.1 615903 59 70 22 0.4 0.1 3.5 630716 57 86 260.5 0.1 3.1 630718 61 72 23 0.4 0.1 3.4 630722 117 153 24 0.4 0.1 3.2630794 90 113 29 0.5 0.1 3.4 630800 92 133 25 0.4 0.1 3.6 630948 48 7721 0.4 0.1 3.3 630950 79 83 25 0.4 0.1 3.3 630952 208 243 31 0.4 0.2 2.9630953 87 135 22 0.4 0.1 3.0 630957 110 115 26 0.4 0.1 3.6 637749 63 10225 0.1 0.2 3.2 647384 135 158 24 0.4 0.1 3.7 647389 243 272 25 0.2 0.23.6 647391 205 520 27 0.0 1.1 2.1 647393 142 172 27 0.2 0.1 3.4 647394391 340 29 0.1 0.2 2.8 647395 68 95 24 0.1 0.1 3.2 647419 53 66 23 0.40.1 3.5 647420 56 80 23 0.1 0.1 3.3 647446 66 110 23 0.2 0.1 3.4 64744754 67 22 0.1 0.1 3.1 647448 55 73 26 0.4 0.1 3.3 647449 46 81 24 0.4 0.13.2 647475 45 78 26 0.4 0.1 3.5 647476 52 85 20 0.4 0.1 3.2 647477 58 8924 0.5 0.1 3.5 647478 50 82.8 22.8 0.4 0.1 3.2 647506 45 95.3 22.9 0.40.1 3.2 647508 73 183.3 33.3 0.3 0.1 2.5 647532 108 179.5 47.8 0.5 0.11.8

TABLE 14 P/C ratio in urine of Sprague-Dawley rats PBS 1.0 585626 6.7585653 9.4 585683 7.0 585698 6.2 585752 13.4 585758 11.5 585774 7.5585775 6.7 585777 7.6 591466 8.0 591514 8.0 591579 7.3 614954 5.2 6158952.9 615897 4.7 615899 4.2 615900 4.5 615903 5.7 630716 3.9 630718 4.5630722 4.3 630794 2.3 630800 5.1 630948 2.4 630950 6.3 630952 6.6 6309534.4 630957 3.8 637749 3.0 647384 2.2 647389 2.4 647391 3.4 647393 3.7647394 9.9 647395 5.2 647419 5.0 647420 4.9 647446 3.8 647447 3.9 6474485.6 647449 5.0 647475 4.1 647476 4.6 647477 5.8 647478 4.6 647506 4.7647508 9.2 647532 49.4

Hematology Assays

Blood samples of approximately 1.3 mL of blood were collected from eachof the available study animals in tubes containing K₂-EDTA and sent toIDEXX Laboratories, Inc. (Fremont, Calif.) for measurement and analysisof red blood cell (RBC) count, white blood cells (WBC) count, individualwhite blood cell counts—such as that of monocytes, neutrophils,lymphocytes—as well as for platelet count, total hemoglobin content andhematocrit (HCT). The results are presented in Table 15. ISISoligonucleotides that caused changes in the levels of any of thehematology markers outside the expected range for antisenseoligonucleotides were excluded in further studies.

TABLE 15 Hematology markers in Sprague-Dawley rats WBC RBC HCTLymphocytes Monocytes Platelets ISIS No. (×10³/μL) (×10⁶/μL) (%) (/mm³)(/mm³) (×10³/μL) PBS 4.8 8.5 52.7 3567 93 812 585626 10.1 8.3 46.9 8969252 1237 585653 13.8 8.2 48.3 11190 359 1305 585683 17.8 7.9 45.7 15773557 826 585698 16.9 7.9 46.0 15380 344 761 585752 15.3 8.0 46.0 11396585 1158 585758 18.4 7.9 44.0 6369 61 1548 585774 14.7 8.5 48.6 12818552 873 585775 7.3 8.4 48.4 6218 219 1161 585777 11.2 8.1 47.1 9548 175982 591466 14.3 8.1 45.6 12519 226 812 591514 14.9 8.5 48.2 10993 1691157 591579 12.5 9.1 51.1 8540 222 1080 614954 13.6 5.2 29.9 12186 441511 615895 15.2 8.0 45.9 11868 603 926 615897 14.5 7.5 43.3 10920 786902 615899 19.8 7.8 43.7 17319 525 566 615900 14.0 7.1 41.0 12167 267770 615903 9.4 8.5 51.3 7113 268 687 630716 21.1 7.8 45.3 18994 449 601630718 8.9 8.9 52.5 7071 269 657 630722 17.0 9.1 51.6 13397 721 693630794 8.8 8.7 50.5 7098 137 529 630800 16.6 8.0 45.3 13210 478 695630948 7.2 8.5 50.2 5359 158 670 630950 11.0 8.8 52.4 8833 307 544630952 24.2 7.7 42.8 17991 798 958 630953 25.0 6.9 42.4 20205 713 662630957 11.7 8.7 50.5 8913 340 684 637749 12.8 7.5 44.7 10837 765 661647384 14.8 9.0 54.5 11682 354 642 647389 12.8 8.2 51.0 10621 534 1075647391 16.8 2.3 20.3 13574 807 240 647393 14.5 6.9 40.8 12467 423 1112647394 24.9 6.5 39.6 21847 1070 990 647395 10.4 7.4 45.2 8685 515 1092647419 13.8 8.3 48.5 11866 257 939 647420 11.1 8.0 47.3 9350 521 1079647446 5.9 7.5 44.8 4805 258 1076 647447 10.2 7.8 47.3 8542 260 1019647448 10.7 7.9 45.3 9050 260 933 647449 21.1 7.7 45.5 18809 479 630647475 17.4 8.3 49.0 14951 562 776 647476 14.2 8.3 47.7 12336 339 979647477 16.8 8.3 46.3 14089 726 697 647478 23.7 7.4 42.9 22039 440 762647506 12.9 7.9 45.4 11679 268 711 647508 12.2 6.8 38.8 9800 431 647647532 33.1 5.3 31.0 27732 963 844

Body and Organ Weights

Body weights of all the groups of rats were measured at the start of theexperiment, and every week until the end of the study. Liver, spleen andkidney weights were also measured at the end of the study, and thechange in body weight and organ weights relative to the PBS controlgroup at baseline are presented in Table 16. ISIS oligonucleotides thatcaused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 16 Body weight and relative organ weights of Sprague-Dawley rats(in grams) at week six ISIS No. Liver (g) Kidney (g) Spleen (g) Bodyweight (g) PBS 1.0 1.0 1.0 1.8 585626 1.1 0.9 2.3 1.4 585653 1.1 1.0 2.11.5 585683 1.1 0.9 3.3 1.4 585698 1.1 0.9 2.8 1.4 585752 1.1 0.9 2.5 1.3585758 1.5 0.9 2.3 1.2 585774 1.1 0.9 2.2 1.4 585775 1.0 0.9 1.7 1.3585777 1.0 0.9 2.3 1.4 591466 1.0 0.9 2.7 1.3 591514 1.1 1.0 2.4 1.1591579 1.0 0.8 1.9 1.3 614954 1.4 1.3 4.1 1.4 615895 1.0 1.1 1.7 1.5615897 1.3 1.1 2.1 1.7 615899 1.1 1.1 2.0 1.6 615900 1.2 1.2 2.1 1.8615903 1.2 1.0 1.5 1.9 630716 1.1 1.1 2.8 1.6 630718 1.1 1.0 2.1 1.8630722 1.2 1.2 1.6 1.5 630794 0.9 1.0 1.6 1.8 630800 1.3 1.3 2.4 1.6630948 1.0 1.1 1.7 1.9 630950 1.2 1.0 2.3 1.8 630952 1.4 1.3 2.6 1.2630953 1.4 1.2 4.2 1.6 630957 1.2 1.0 1.7 1.6 637749 1.4 1.3 4.4 1.4647384 1.0 1.0 1.1 1.7 647389 1.0 1.1 1.8 1.7 647391 1.8 1.5 13.1 1.4647393 1.3 1.1 1.8 1.6 647394 1.2 1.2 2.8 1.6 647395 1.3 1.3 1.8 1.7647419 1.3 1.1 1.6 1.8 647420 1.2 1.1 2.1 1.6 647446 1.3 1.2 2.3 1.8647447 1.1 1.1 1.9 1.7 647448 1.2 1.2 1.6 1.7 647449 1.2 1.2 1.7 1.7647475 1.2 1.1 1.5 1.7 647476 1.1 1.1 1.5 1.5 647477 1.2 1.1 1.7 1.6647478 1.2 1.3 1.8 1.7 647506 1.2 1.3 2.0 1.6 647508 1.7 2.1 2.9 1.3647532 2.0 1.7 3.7 1.3

Example 6: Effect of Antisense Inhibition of TMPRSS6 in Transgenic MouseModel

About 32 antisense oligonucleotides found tolerable in the rat studiesabove were further evaluated for their ability to reduce human TMPRSS6mRNA transcript in mice with the human TMPRSS6 transgene (“huTMPRSS6” or“Tg” mice).

Treatment

Eight to sixteen week old male and female huTMPRSS6 transgenic mice wereinjected subcutaneously with five doses of 6 mg/kg per dose of ISISantisense oligonucleotides targeting TMPRSS6, administered over a periodof two weeks (30 mg/kg total), or with PBS as a control. Each treatmentgroup consisted of 4 animals. Forty-eight hours after the administrationof the last dose, blood was drawn from each mouse and the mice weresacrificed and tissues were collected.

RNA Analysis

At the end of the study, RNA was extracted from liver for real-time PCRanalysis of liver TMPRSS6 mRNA expression. Results are presented inTable 17 as percent inhibition with respect to PBS treated animals.Human primer probe set RTS4586 (forward sequence TGATAACAGCTGCCCACTG,designated herein as SEQ ID NO: 86; reverse sequenceTCACCTTGAAGGACACCTCT, designated herein as SEQ ID NO: 87; probe sequenceAGTITCTGCCACACCTITGCCCA, designated herein as SEQ ID NO: 88) was used tomeasure mRNA levels. The mRNA levels were normalized with levels ofcyclophilin A, a housekeeping gene, which were determined using primerprobe set mCYCLO_24 (forward primer TCGCCGCTTGCTGCA, designated hereinas SEQ ID NO: 89; reverse primer ATCGGCCGTGATGTCGA, designated herein asSEQ ID NO: 90; probe CCATGGTCAACCCCACCGTGTTC, designated herein as SEQID NO: 91).

TABLE 17 % inhibition of TMPRSS6 mRNA in transgenic mice livernormalized to PBS expression % ISIS No inhibition 585626 57 585653 74585683 81 585698 59 585698 59 585774 69 585775 81 591514 73 615899 88615900 88 615903 97 630716 82 630718 99 630722 92 630794 71 630800 81630948 65 630950 81 630957 70 647384 66 647393 95 647395 100 647419 99647420 96 647446 84 647447 89 647448 96 647449 88 647475 84 647476 84647477 96 647478 91 647506 91

Example 7: Antisense Compounds Conjugated to GalNAc₃ Targeting TMPRSS6

The sequences of selected antisense oligonucleotides targeting TMPRSS6found potent and tolerable in the examples above were chosen as parentsequences to design new GalNAc₃ conjugated antisense compounds targetinghuman TMPRSS6.

As summarized in Table 18, below, each of the newly designed antisensecompounds described in this example had a 5′-Trishexylamino-(THA)-C6GalNAc₃ endcap. ISIS 702843 was a 5-10-5 MOE gapmer having a mixed(phosphorothioate and phosphodiester) backbone (“MBB”) with a5′-Trishexylamino-(THA)-C6 GalNAc₃ endcap. ISIS 705051, 705052 and705053 were 5-10-5 MOE gapmers having a phosphorothioate backbone with a5′-Trishexylamino-(THA)-C6 GalNAc₃ endcap. ISIS 706940 was a 3-10-3 cEtgapmer with all phosphorothioate internucleoside linkages and a5′-Trishexylamino-(THA)-C6 GalNAc₃ endcap; ISIS 706941, 706942 and706943 are deoxy, MOE, and (S)-cEt containing gapmers having aphosphorothioate backbone with a 5′-Trishexylamino-(THA)-C6 GalNAc₃endcap.

TABLE 18 Eight unconjugated antisense compounds targeting TMPRSS6mRNA and corresponding GalNAc₃ conjugate antisense compounds ParentGalNAc Sequence Conjugated SEQ ISIS# ISIS# Backbone Length SequenceChemistry ID NO 585774 702843 MBB 20 CTTTATTCCAAAGGGCAGCT 5′-THA GalNAc₃36 5-10-5 MOE 585774 705051 PS 20 CTTTATTCCAAAGGGCAGCT 5′-THA GalNAc₃ 365-10-5 MOE 585683 705052 PS 20 GCACGGCAAATCATACTTCT 5′-THA GalNAc₃ 235-10-5 MOE 585775 705053 PS 20 AGCTTTATTCCAAAGGGCAG 5′-THA GalNAc₃ 375-10-5 MOE 630718 706940 PS 16 AGCTTTATTCCAAAGG 5′-THA GalNAc₃ 63kkk-d10-kkk 647477 706941 PS 16 AGCTTTATTCCAAAGG 5′-THA GalNAc₃ 63kk-d8-eeeekk 647449 706942 PS 16 CAGCTTTATTCCAAAG 5′-THA GalNAc₃ 77kk-d9-eeekk 647420 706943 PS 16 CAGCTTTATTCCAAAG 5′-THA GalNAc₃ 77kek-d9-eekk

All of the oligonucleotides sequences described in Table 18 werecomplementary to both human and Rhesus monkey sequences. At the time thestudies described herein were undertaken, the cynomolgus monkey genomicsequence for TMPRSS6 was not available in the National Center forBiotechnology Information (NCBI) database; therefore, cross-reactivityof antisense oligonucleotides targeting human TMPRSS6 with thecynomolgus monkey gene sequence could not be confirmed. Instead, thesequences of antisense oligonucleotides were compared to a rhesus monkeysequence for homology. It is expected that ISIS oligonucleotides withhomology to the rhesus monkey sequence are fully cross-reactive with thecynomolgus monkey sequence as well.

The antisense oligonucleotides selected for GalNAc conjugation are fullycomplementary to the rhesus genomic sequence (the complement of GENBANKAccession No. NW_001095180.1, truncated from nucleotides 380000 to422000, designated herein as SEQ ID NO: 95). The start and stop sites ofeach oligonucleotide to the rhesus sequence is presented in Table 19while the start and stop sites of each oligonucleotide to the humansequence is presented in Table 20. “Start site” indicates the 5′-mostnucleotide to which the gapmer is targeted in the rhesus monkey or humansequences.

TABLE 19 ASOs complementary to the rhesus TMPRSS6 genomic sequence(SEQ ID NO: 95) rhesus rhesus SEQ ISIS Start Stop ID No Site SiteChemistry Sequence NO 585774 40518 40537 5-10-5 MOE CTTTATTCCAAAGGGCAGCT36 702843 40518 40537 5′-THA GalNAc₃ 5-10-5 MOE CTTTATTCCAAAGGGCAGCT 36705051 40518 40537 5′-THA GalNAc₃ 5-10-5 MOE CTTTATTCCAAAGGGCAGCT 36705052 22499 22518 5′-THA GalNAc₃ 5-10-5 MOE GCACGGCAAATCATACTTCT 23705053 40520 40539 5′-THA GalNAc₃ 5-10-5 MOE AGCTTTATTCCAAAGGGCAG 37630718 40524 40539 kkk-10-kkk AGCTTTATTCCAAAGG 63 706940 40524 405395′-THA GalNAc₃ kkk-10-kkk AGCTTTATTCCAAAGG 63 706941 40524 405395′-THA GalNAc₃ kk-8-eeeekk AGCTTTATTCCAAAGG 63 706942 40525 405405′-THA GalNAc₃ kk-9-eeekk CAGCTTTATTCCAAAG 77 706943 40525 405405′-THA GalNAc₃ kk-9-eeekk CAGCTTTATTCCAAAG 77

TABLE 20 Sites on TMPRSS6 mRNA (SEQ ID NO: 1) and/or genomic (SEQ ID NO:2) sequences targeted by GalNAc₃- modified antisense oligonucleotidesSEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ ISIS Start StopStart Stop ID NO Site Site Site Site NO 702843 3162 3181 44924 44943 36705051 3162 3181 44924 44943 36 705052 1286 1305 26046 26065 23 7050533164 3183 44926 44945 37 706940 3168 3183 44930 44945 63 706941 31683183 44930 44945 63 706942 3169 3184 44931 44946 77 706943 3169 318444931 44946 77

Example 8: Tolerability of GalNAc3-Modified Antisense OligonucleotidesTargeted to Human TMPRSS6 in CD-1 Mice

CD1® mice (Charles River, Mass.) were treated with ISIS GalNAc₃-modifiedantisense oligonucleotides described in Table 18 above, and evaluatedfor changes in the levels of various plasma chemistry markers.

Treatment

Groups of six-week-old male CD1 mice (n=4 per treatment group) wereinjected subcutaneously twice a week for six weeks with 40 mg/kg of ISISMOE gapmer GalNAc3-modified antisense oligonucleotides (80 mg/kg/weekdose) or with 20 mg/kg of ISIS (S)-cEt containing gapmerGalNAc3-modified antisense oligonucleotides described in Table 14 above(40 mg/kg/week dose). One group of male CD1 mice was injectedsubcutaneously twice a week for 6 weeks with PBS. Mice were euthanized48 hours after the last dose, and organs and plasma were harvested forfurther analysis. Liver, kidney, spleen, heart and lung were collectedfor histology, and plasma was collected to measure levels of certainplasma chemistry markers.

Plasma Chemistry Markers

To evaluate the effect of ISIS GalNAc₃-modified antisenseoligonucleotides on liver and kidney function, plasma levels oftransaminases, bilirubin, albumin, creatinine, and BUN were measuredusing an automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). The results are presented in Table 21. ISISoligonucleotides causing changes in the levels of any of the liver orkidney function markers outside the expected range for antisenseoligonucleotides were excluded from further studies.

TABLE 21 Plasma chemistry markers in CD1 mice at week six ALT AST BUNCreat Tbil Alb ISIS No. (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) (g/dL) PBS32 70 27.3 0.12 0.17 2.8 702843 59 72 28 0.17 0.16 2.9 705051 47 73 26.60.16 0.17 2.8 705052 81 94 26.3 0.16 0.17 2.8 705053 139 129 28.2 0.170.18 2.9 706940 46 66 28.1 0.18 0.14 3.0 706941 40 57 25.5 0.18 0.16 2.9706942 195 145 27 0.16 0.14 3.0 706943 178 144 26.1 0.16 0.16 3.9

Body and Organ Weights

Body weights of all groups of mice were measured at the start of theexperiment, and every week until the end of the study. Liver, kidney andspleen weights were also measured at the end of the study, and thechange in body weight and organ weights relative to the PBS controlgroup at baseline are presented in Table 22. ISIS oligonucleotides thatcaused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 22 Change in body weight and relative organ weights of CD1 mice(in grams) at week six BW change Relative liver Relative kidney Relativespleen ISIS No. (g) weight (g) weight (g) weight (g) PBS 1.41 1.00 1.001.00 702843 1.39 1.05 1.00 1.08 705051 1.38 0.98 1.00 1.05 705052 1.391.02 0.96 1.32 705053 1.37 1.03 0.98 1.22 706940 1.31 0.97 1.01 1.16706941 1.39 0.90 0.98 1.12 706942 1.39 1.09 1.09 1.40 706943 1.44 1.061.02 1.08

Hematology

To evaluate any effect of ISIS GalNAc₃-modified antisenseoligonucleotides in CD1 mice on hematologic parameters, blood samples ofapproximately 1.3 mL of blood was collected from each of the availablestudy animals in tubes containing K₂-EDTA. Samples were analyzed for redblood cell (RBC) count, white blood cells (WBC) count, individual whiteblood cell counts, such as that of monocytes, neutrophils, lymphocytes,as well as for platelet count, hemoglobin content and hematocrit, usingan ADVIA120 hematology analyzer (Bayer, USA). The data is presented inTable 23.

The data indicate the oligonucleotides did not cause significant changesin hematologic parameters outside the expected range for antisenseoligonucleotides at this dose. Generally, ISIS GalNAc-conjugatedantisense oligonucleotides were well tolerated in terms of thehematologic parameters of the mice.

TABLE 23 Blood cell counts in CD1 mice WBC RBC Platelets (×10³/ (×10⁶/HCT Lymphocytes Monocytes (×10³/ ISIS No. μL) μL) (%) (/mm³) (/mm³) μL)PBS 2.9 8.9 49.9 1916.5 38.8 659.0 702843 4.9 8.9 48.5 3630.0 90.3 700.5705051 4.0 8.5 47.8 2961.0 80.7 781.3 705052 3.2 9.3 50.7 2553.7 146.0750.7 705053 5.3 9.1 49.8 3856.0 179.5 913.3 706940 3.7 8.5 46.7 2591.3154.0 935.3 706941 5.5 8.8 49.9 3940.3 177.5 911.8 706942 5.7 9.4 51.84126.3 155.3 955.7 706943 3.4 8.9 48.2 3067.0 0.0 1021.3

Histological assessment of the GalNAc-conjugated TMPRSS6 antisensecompounds in liver, spleen, kidney, heart and lung from the CD-1 Micewas performed. Overall, despite dosing GalNAc₃-conjugated antisenseoligonucleotides at doses having approximately 8-times more activity inliver than unconjugated oligonucleotides, they were well tolerated anduseful compounds for inhibiting TMPRSS6 and are important candidates forthe treatment of an iron accumulation disease, disorder or condition.

Example 9: Dose-Response of Antisense Oligonucleotides Targeting TMPRSS6in huTMPRSS6 Transgenic Mice

The eight ISIS GalNAc₃-modified antisense oligonucleotides targetingTMPRSS6 (ISIS Nos. 702843, 705051, 705052, 705053, 706940, 706941,706942 and 706943) as well as two parent compounds (ISIS 585774 and ISIS630718) were tested and evaluated in a dose-response study for theirability to inhibit human TMPRSS6 mRNA expression in huTMPRSS6 transgenicmice.

Treatment

huTMPRSS6 Tg mice were maintained on a 12-hour light/dark cycle and werefed ad libitum normal mouse chow. Animals were acclimated for at least 7days in the research facility before initiation of the experiment.Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS)and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

Male and female huTMPRSS6 mice, roughly 3.5 to 4.5 months old, weredivided into 44 groups of four mice each (two males and two females ineach group). The mice received subcutaneous injections of ISISoligonucleotide, twice per week for three weeks. One group of micereceived subcutaneous injections of PBS twice per week for three weeks.Forty-eight hours after the administration of the last dose, blood wasdrawn from each mouse and the mice were sacrificed and tissues werecollected.

RNA Analysis

At the end of the treatment period, total RNA was extracted from thelivers of transgenic mice for quantitative real-time PCR analysis andmeasurement of human TMPRSS6 mRNA expression. TMPRSS6 mRNA levels werenormalized with levels of cyclophilin A, a housekeeping gene, which weredetermined using mCYCLO_24 primer probe set according to standardprotocols. The results below are presented in Table 24 as the averagepercent of TMPRSS6 mRNA levels for each treatment group, normalized toPBS-treated control and are denoted as “% PBS”. Values above 100 weresimply noted as “100”. Negative values were simply noted as “0”.

Human primer probe set RTS4586 (forward sequence TGATAACAGCTGCCCACTG,designated herein as SEQ ID NO: 86; reverse sequenceTCACCTTGAAGGACACCTCT, designated herein as SEQ ID NO: 87; probe sequenceAGTTCTGCCACACCTTGCCCA, designated herein as SEQ ID NO: 88) was used tomeasure mRNA levels.

TABLE 24 Response to eight ISIS GalNAc₃-conjugated and two unconjugatedcompounds targeting TMPRSS6 in Tg mice Dose TMPRSS6 TMPRSS6 Treatment(mpk/wk) % PBS % Inhibition 585774 100 4 96 30 35 65 10 99 1 3 100 0702843 10 0 100 3 16 84 1 55 45 0.3 100 0 705051 10 1 99 3 68 32 1 72 280.3 100 0 705052 10 28 72 3 23 77 1 100 0 0.3 100 0 705053 10 7 93 3 3070 1 100 0 0.3 100 0 630718 30 0 100 10 37 63 3 100 0 1 100 0 706940 3 0100 1 4 96 0.3 52 48 0.1 100 0 706941 3 8 92 1 71 29 0.3 100 0 0.1 100 0706942 3 2 98 1 47 53 0.3 82 18 0.1 100 0 706943 3 2 98 1 15 85 0.3 1000 0.1 100 0

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, serum levels of transaminases, biliribin and BUN were measuredusing an automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.) and presented in Table 25 below. ISIS oligonucleotidescausing changes in the levels of any of the liver or kidney functionmarkers outside the expected range for antisense oligonucleotides wereexcluded from further studies.

TABLE 25 Serum chemistries of eight ISIS GalNAc₃-modified ASOs and twounconjugated compounds targeting TMPRSS6 in transgenic mice Dose(mg/kg/wk) ALT AST BUN PBS n/a 39.5 64.8 40.4 585774 100 40.8 68.5 42.530 36.5 70.8 37.2 10 38.5 59.0 38.9 3 39.8 59.5 41.6 702843 10 38.3 57.335.6 3 41.8 65.5 38.9 1 41.8 100.3 34.7 0.3 43.3 65.3 38.8 705051 1047.3 79.8 35.4 3 37.0 58.5 34.9 1 33.0 57.0 35.7 0.3 42.0 67.5 34.6705052 10 34.8 61.5 33.9 3 37.0 62.5 32.8 1 35.8 57.8 35.1 0.3 35.0 65.034.1 705053 10 39.0 55.8 32.4 3 35.3 62.8 38.6 1 39.8 73.5 36.6 0.3 39.573.3 37.9 630718 30 58.8 160.8 37.7 10 38.3 73.0 33.8 3 39.3 92.3 32.8 138.0 67.8 35.0 706940 3 36.3 54.8 33.7 1 39.8 65.0 35.7 0.3 38.3 66.834.9 0.1 36.8 52.8 31.8 706941 3 37.5 59.0 31.6 1 34.3 75.8 32.3 0.340.5 72.8 34.9 0.1 45.3 63.8 31.3 706942 3 34.3 90.5 35.8 1 36.8 58.332.8 0.3 46.8 270.0 39.8 0.1 35.5 76.5 31.0 706943 3 35.5 81.3 34.6 133.3 71.8 31.0 0.3 35.0 54.5 32.2 0.1 42.3 60.0 33.1

All GalNAc conjugated ASOs were well-tolerated with no major changes inorgan and body weights nor serum transaminase levels.

The half maximal effective dosage (ED₅₀) of each ASO was calculated andis presented in Table 26, below.

TABLE 26 Potencies of eight ISIS GalNAc₃- modified ASOs and twounconjugated compounds targeting TMPRSS6 ISIS # ED₅₀ (mpk/wk) 58577426.0 702843 ~1.0 705051 3.7 705052 ~2.7 705053 ~2.8 630718 ~9.7 706940~0.3 706941 1.3 706942 0.9 706943 ~0.9

ED₅₀ calculations showed that GalNAc-conjugated ASOs are approximately10-fold more potent than unconjugated ASOs. ISIS 702843 was the mostpotent GalNAc conjugated 5-10-5 MOE gapmer compound.

Example 10: Viscosity Assessment of Antisense Oligonucleotides TargetingTMPRSS6

The viscosity of the antisense oligonucleotides was measured with theaim of screening out antisense oligonucleotides which have a viscositymore than 40 cP. Oligonucleotides having a viscosity greater than 40 cPwould not be optimal for administration to a subject.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μLof water was added and the antisense oligonucleotide was dissolved intosolution by heating the vial at 50° C. Part of (75 μL) the pre-heatedsample was pipetted to a micro-viscometer (Cambridge). The temperatureof the micro-viscometter was set to 25° C. and the viscosity of thesample was measured. Another part (20 μL) of the pre-heated sample waspipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UVinstrument). The results are presented in Table 27 and indicate thatmost of the GalNAc antisense oligonucleotides solutions are optimal intheir viscosity under the criterion stated above. Antisenseoligonucleotide 706941 was the only antisense oligonucleotide testedthat had a viscosity level above 40 cP.

TABLE 27 Viscosity Data for GalNAc-Conjugated ASOs ISIS # Chemistry cP702843 5′-THA GalNAc₃ 5-10-5 MOE (MBB) 33 705051 5′-THA GalNAc₃ 5-10-5MOE (PS) 23 705052 5′-THA GalNAc₃ 5-10-5 MOE (PS) 16 705053 5′-THAGalNAc₃ 5-10-5 MOE (PS) 26 706940 5′-THA GalNAc₃ kkk-10-kkk (PS) 39706941 5′-THA GalNAc₃ kk-8-eeeekk (PS) 54 706942 5′-THA GalNAc₃kk-9-eeekk (PS) 20 706943 5′-THA GalNAc₃ kek-9-eekk (PS) 19

Example 11: Antisense Inhibition In Vivo by Oligonucleotides TargetingTMPRSS6 Comprising a GalNAc₃ Conjugate in Cynomolgus Monkeys

At the time this study was undertaken, the cynomolgus monkey genomicsequence for TMPRSS6 was not available in the National Center forBiotechnology Information (NCBI) database; therefore, cross-reactivityof antisense oligonucleotides targeting human TMPRSS6 with thecynomolgus monkey gene sequence could not be confirmed. Instead, thesequences of antisense oligonucleotides were compared to a rhesus monkeysequence for homology as described in Example 6, above. It is expectedthat ISIS oligonucleotides with homology to the rhesus monkey sequenceare fully cross-reactive with the cynomolgus monkey sequence as well.

The ten human TMPRSS6 antisense oligonucleotides selected for testing incynomolgus monkey had 0 mismatches with the rhesus genomic sequence (SEQID NO: 95) as described in Example 6, above.

Study Design

Ten antisense oligonucleotides were evaluated for efficacy andtolerability, and for their pharmacokinetic profile in the liver andkidney in a 13-week study of antisense inhibition of TMPRSS6 mRNA inmale cynomolgus monkeys. The monkeys were treated by subcutaneousadministration with the eight ISIS GalNAc₃-modified ASOs and twounconjugated parent antisense oligonucleotides antisenseoligonucleotides targeting TMPRSS6 as shown in Table 28.

TABLE 28 ASOs compared in cynomolgus monkey studies Group ISIS# Dose  1PBS Control n/a  2 585774 25 mpk  3 705051 30 mpk  4 705052 30 mpk  5705053 30 mpk  6 702843 30 mpk  7 705051  5 mpk  8 702843  5 mpk  9630718 23 mpk 10 706940 30 mpk 11 706941 30 mpk 12 706942 30 mpk 13706943 30 mpk 14 706940  5 mpk

High-dose (30mpk) groups for the GalNAc-conjugated ASOs assessedtoxicity. Low-dose (5 mpk) groups for GalNAc-conjugated ASOs werecompared to a corresponding unconjugated parent sequence to assessactivity. Groups 2, 3, 6, 7 and 8 are the same sequence, and the mixedbackbone (MBB) compound ISIS No. 702843 is tested at both low and highdoses, as well as compared to the full phosphorothioate compound ISISNo. 705051 (also tested at both low and high doses). Groups 9, 10, 11and 14 are the same sequence, and ISIS No. 706940 is tested at both lowand high doses.

Treatment

Prior to the study, the monkeys were kept in quarantine during which theanimals were observed daily for general health. The monkeys were two tofour years old and weighed two to four kg. 56 male cynomolgus monkeyswere randomly assigned to 14 treatment groups with four monkeys pergroup. Monkeys were each injected subcutaneously every other day for thefirst week, and then once weekly for 11 weeks for a total of 15 doseswith ISIS oligonucleotide or PBS using a stainless steel dosing needleand syringe of appropriate size. Tail bleeds were conducted at 1 weekprior to the first administration, then again at days 9, 16, 30, 44, 58,72 and 86.

During the study period, the monkeys were observed twice daily for signsof illness or distress. Any animal experiencing more than momentary orslight pain or distress due to the treatment, injury or illness wastreated by the veterinary staff with approved analgesics or agents torelieve the pain after consultation with the Study Director. Any animalin poor health or in a possible moribund condition was identified forfurther monitoring and possible euthanasia. Scheduled euthanasia of theanimals was conducted on day 86. The protocols described in the Examplewere approved by the Institutional Animal Care and Use Committee(IACUC).

Prior to the first dose and at various time points thereafter, blooddraws were performed for clinical pathology endpoints (hematology,clinical chemistry, coagulation, Complement Bb and C3, cytokine andchemokine analyses), and urine chemistry was also measured. At baselineand at the end of the experimental period, certain pharmacologyendpoints were measured, such as liver TMPRSS6 mRNA expression, serumhepcidin (Intrinsic LifeSciences, San Diego, Calif.), serum iron andserum transferrin saturation. At the end of the study, body and organweights, histopathology of tissues and PK analysis of liver and kidneywere measured. No significant changes in body weight, cytokine oralbumin levels were observed.

TMPRSS6 RNA Analysis

At the end of the study, RNA was extracted from liver for real-time PCRanalysis of measurement of mRNA expression of TMPRSS6 using variousprimer-probe sets. Representative data using the primer probe setRTS3840 is presented in the table below. Results in Table 29 arepresented as percent inhibition of TMPRSS6 mRNA relative to salinecontrol, normalized with cyclophilin (mCYCLO_24 primer probe set).

TABLE 29 Reduction of monkey liver TMPRSS6 mRNA after 12-weeks ASOadministration % Treatment Dose (mg/kg) inhibition Group 585774 25 76 2705051 30 90 3 705052 30 64 4 705053 30 49 5 702843 30 89 6 705051 5 777 702843 5 82 8 630718 23 65 9 706940 30 71 10 706941 30 72 11 706942 3093 12 706943 30 91 13 706940 5 61 14

ISIS Nos. 705051, 702843, 706942 and 706943 were quite efficacious,demonstrating ≥89% target reduction at 30 mpk after 13-weeks of dosing.

Hepcidin Analysis

Serum hepcidin levels were measured at the time points shown in Table 30below. Results are presented as percent saline control. “Day −7”indicates one week before the first dose was administered.

TABLE 30 Monkey serum hepcidin levels Dose (mg/kg) Day −7 Day 9 Day 16Day 44 Day 86 Saline n/a 1.0 1.0 1.0 1.0 1.0 585774 25 0.9 1.3 1.4 1.11.4 705051 30 0.9 1.1 1.5 1.5 1.8 702843 30 0.9 1.2 1.2 1.3 1.9 70694230 0.7 1.0 1.5 1.3 1.9 706943 30 0.8 0.9 1.5 1.2 1.6

The table shows that serum hepcidin levels increased over the course ofthe study.

Serum Iron and Transferrin Saturation Analysis

The averages of the four subjects from each of the 14 treatment groupsare presented in Table 31, below. As is shown in Table 31, serum ironlevels and transferrin saturation (“Tf sat”) were reduced at day 86 intreated groups compared to control.

TABLE 31 Monkey serum iron and transferrin saturation levels at day 86Group Dose # Treatment (mg/kg) iron Tf sat  1 Saline n/a 125.7 38.8  2585774 25 55.2 15.7  3 705051 30 36.6 10.0  4 705052 30 61.9 15.8  5705053 30 96.0 27.0  6 702843 30 42.3 13.3  7 705051 5 63.7 20.0  8702843 5 51.7 16.5  9 630718 23 61.4 17.7 10 706940 30 71.6 20.5 11706941 30 55.7 15.8 12 706942 30 25.9 6.9 13 706943 30 30.3 7.4 14706940 5 82.8 23.7

1-36. (canceled)
 37. An oligomeric compound according to the followingchemical structure:

or a salt thereof.
 38. The oligomeric compound of claim 37, which is thesodium salt or the potassium salt.
 39. An oligomeric compound accordingto the following chemical structure:


40. An oligomeric compound, wherein the anion form of the oligomericcompound has the following chemical structure:


41. An oligomeric compound comprising a modified oligonucleotideaccording to the following formula: THA-C6 GalNAc₃ mCks Aes Gks mCds TdsTds Tds Ads Tds Tds mCds mCds Aes Aes Aks Gk (SEQ ID NO: 77); wherein,A=an adenine nucleobase, mC=a 5-methylcytosine nucleobase, G=a guaninenucleobase, T=a thymine nucleobase, e=a 2′-MOE sugar moiety, d=a2′-β-D-deoxyribosyl sugar moiety, s=a phosphorothioate internucleosidelinkage, o=a phosphodiester internucleoside linkage, k=a cEt sugarmoiety, and THA-C6 GalNAc₃=

wherein the cleavable moiety (CM) comprises a phosphodiester bond.
 42. Apopulation of oligomeric compounds of claim 37, wherein all of thephosphorothioate internucleoside linkages of the modifiedoligonucleotides are stereorandom.
 43. A pharmaceutical compositioncomprising an oligomeric compound of claim 37 and a pharmaceuticallyacceptable diluent.
 44. The pharmaceutical composition of claim 43,wherein the pharmaceutically acceptable diluent is phosphate-bufferedsaline (PBS) or water.
 45. A population of oligomeric compounds of claim38, wherein all of the phosphorothioate internucleoside linkages of themodified oligonucleotides are stereorandom.
 46. A pharmaceuticalcomposition comprising an oligomeric compound of claim 38 and apharmaceutically acceptable diluent.
 47. The pharmaceutical compositionof claim 46, wherein the pharmaceutically acceptable diluent isphosphate-buffered saline (PBS) or water.
 48. A population of oligomericcompounds of claim 39, wherein all of the phosphorothioateinternucleoside linkages of the modified oligonucleotides arestereorandom.
 49. A pharmaceutical composition comprising an oligomericcompound of claim 39 and a pharmaceutically acceptable diluent.
 50. Thepharmaceutical composition of claim 49, wherein the pharmaceuticallyacceptable diluent is phosphate-buffered saline (PBS) or water.
 51. Apopulation of oligomeric compounds of claim 40, wherein all of thephosphorothioate internucleoside linkages of the modifiedoligonucleotide are stereorandom.
 52. A pharmaceutical compositioncomprising the oligomeric compound of claim 40 and a pharmaceuticallyacceptable diluent.
 53. The pharmaceutical composition of claim 52,wherein the pharmaceutically acceptable diluent is phosphate-bufferedsaline (PBS) or water.
 54. A population of oligomeric compounds of claim41, wherein all of the phosphorothioate internucleoside linkages of themodified oligonucleotide are stereorandom.
 55. A pharmaceuticalcomposition comprising the oligomeric compound of claim 41 and apharmaceutically acceptable diluent.
 56. The pharmaceutical compositionof claim 55, wherein the pharmaceutically acceptable diluent isphosphate-buffered saline (PBS) or water.
 57. A method of reducingexpression of TMPRSS6 in a cell comprising contacting the cell with anoligomeric compound of claim
 37. 58. A method comprising administeringthe compound of claim 37 to a subject in need thereof.
 59. The method ofclaim 58, wherein the subject has polycythemia, hemochromatosis oranemia.
 60. The method of claim 59, wherein the anemia is β-thalassemia.61. The method of claim 59, wherein the polycythemia is polycythemiavera.
 62. The method of claim 58, wherein the subject is human.